Modelling trigeminovascular pain in the unrestrained rat: an … · 2016-03-08 · of migraine with aura.353,363 The median attack frequency in active migraineurs ranges from 0.4352

Modelling trigeminovascular pain in the

unrestrained rat: an approach to a better

understanding of migraine headache

ii

The study described in this thesis was carried out at the Departments of Anaestesiology and

Biological Psychiatry, University Hospital Groningen, the Netherlands, within the framework

of the School of Behavioral, Cognitive and Neurosciences. The work was generously

supported by Glaxo-Wellcome, Zeist, The Netherlands.

Financial support by Glaxo-Wellcome for publication of this thesis is gratefully

acknowledged.

ISBN 90-367-1173-8

© by Richard Kemper, Zwolle, the Netherlands, 1999.

Printed by Stichting Drukkerij C. Regenboog, Groningen, The Netherlands

RIJKSUNIVERSITEIT GRONINGEN

Modelling trigeminovascular pain in the unrestrained rat: an approach toa better understanding of migraine headache

Proefschrift

ter verkrijging van het doctoraat in deMedische Wetenschappen

aan de Rijksuniversiteit Groningenop gezag van de

Rector Magnificus, dr. D.F.J. Bosscher,in het openbaar te verdedigen op

woensdag 12 januari 2000om 16.00 uur

door

Richard Hendrikus Antonius Kemper

geboren op 22 januari 1971te Kampen

iv

Promotor: Prof. dr. J. KorfCo-promotor: Dr. G.J. ter HorstReferent: Dr. W.J. Meijler

v

Promotiecommissie: Prof. dr. J.A. de Boer, Rijksuniversiteit GroningenProf. dr. P.G.M. Luiten, Rijksuniversiteit GroningenProf. dr. J. Schoenen, University Liège, Belgium

Aan mijn ouders

vi

vii

Contents

Section 1: Introduction 9Migraine history 10Migraine present time 12Migraine pathophysiology 13Animal models of migraine headache 19Aim and outline of this thesis 23Section 2: Characterization of an animal model of trigeminovascular

headache in the unrestrained rat 25Preface 26Chapter 2.1: Trigeminovascular stimulation in conscious rats 27Chapter 2.2: Patterns of cerebral activation associatied with headache in the conscious

rat; a Fos-immunoreactivity studie 35Section 3: Immunesystem modulation of trigeminovascular headache 53Preface 54Chapter 3.1: Immune system function in migraine; a review 55Chapter 3.2: Lipopolysaccharide-induced hyperalgesia of intracranial capsaicin sensitive

afferents in conscious rats 75Section 4: Central pharmacological modulation of trigeminovascular

headache 93Preface 94Chapter 4.1: Intracisternally applied octreotide does not ameliorate trigeminovascular

nociception 95Chapter 4.2: Neuronal nitric oxide synthase inhibition in acute trigeminovascular Nociception 107Section 5: General Discussion 117Summary of the results 118Conscious vs. anaesthetized 118Behaviour in-depth 119Peripheral vs. central 120References 123Samenvatting 147Dankwoord 152Curriculum Vitae 157

viii

Section 1

Introduction

Section 1

10

Migraine historyMigraine has been known about for a long time already. From the days that man

could write, descriptions are present that hint of migraine. Alvarez5 discovered a description

written, in a poem in Sumeria in Mesopotamia 3,000 years before Christ, where the poet

says:“The sick eyed says not 'l am sick eyed',

The sick-headed (says) not 'I am sick-headed.’”

Another poet from ancient Mesopotamia wrote (see5):"The head throbs,When pain smites the eyesAnd vision is dimmed."

Nowadays, a general practitioner would immediately think of a migraine if a patient were to

complain of disturbed vision in combination with a throbbing headache behind the eyes.

Somewhat later, 3,500 years ago, the oldest known complete medical book, theEbers papyrus found in the Tomb of Thebes, Egypt, mentions “a sickness of half of thehead” referring to the unilateral nature of migraine.

The typical aura and the observation that the headache commences after the aurastops, was first described by Hippocrates (400 AD): ‘... He seemed to see something shiningbefore him like a light, usually in part of the right eye; at the end of a moment, a violentpain supervened in the right temple, then in all the head and neck...’, who also observedthat the headache could be relieved by vomiting. Better descriptions of migraine

characteristics were found later on. Celsus (25 BC to AD 50) was the first to indicate that

migraine was a life-long non-fatal disorder, that there were trigger factors and alsoemphasized that the headache could be localized or generalized: ‘A long weakness of thehead, but neither severe nor dangerous, through the whole life. Sometimes the pain is moreviolent, but short, yet not fatal; which is contracted either by drinking wine, or crudity, orcold, or heat of a fire, or the sun ... Sometimes they afflict the whole head, at other times apart of it’ (see359).

Soranus of Ephesus (AD 90-138) and Aretaeus of Cappadocia (AD 30-90) bothrecognize the combination of a unilateral headache (‘the pain... remains in the half of thehead’) with nausea and vomiting. Aretaeus of Cappadocia also noted photo and

phonophobia (‘For they flee the light; the darkness soothes their disease; nor can they bearreadily to look upon or hear anything disagreeable’). He also introduced the earliest known

comprehensive classification of primary headaches, and separated migraine (‘heterocrania’ –

unilateral, blackness before eyes, nausea, photophobia) from cephalalgia (not very severe,short-lasting) and cephalea (instense, chronic, frequent) (see165,359). Concerning

heterocrania, he said that if they begin at dusk, they end by midday on the next day, and if

they begin at midday, they end by nightfall. "It is rare for the attack to last longer." Aboutthe same time, Galen (AD 131-201) introduced the term "hemicrania", which was later

Introduction

11

modified gradually from hemigrania, emigrania, migrania, megrim, to its present form,

migraine (see359).In his ‘Practice of Physick’ which was published posthumous in 1684, Thomas Willis

(1621-1675) hypothesized that intracranial vasodilatation caused the headache of migraine.

Latham (1872), later argued that visual auras are caused by contraction of the cerebralarteries (see94)

Levine’s ‘On Megrim, Sick-Headache, and Some Allied Disorders’ was published in

1873. Herein Levine describes many individual migraine patients, he recognizes theenormous variety of migraine forms and especially put forward the theory that migraine is

part of a continuum of paroxysmal disorders that is characterized by nerve storms.239

The first description of effective pharmacological treatment of migraine withergotamine tartrate, which is still used by many migraineurs today, was reported in 1929.453

A great contribution to the discovery of the pathophysiological mechanisms causing

migraine was made by Wolff and colleagues. They observed that amyl nitrite in doses that

Sumeria, Mesopotamia. First descriptions that hint of migraine

Thomb of Thebes, Egypt. Ebers Papyrus mentions 'sickness of half of the head'

Celsus: Migraine is life-long, non-fatal, has trigger factors and can be localized or generalized

Aretaeus of Cappadocia: Introduced term 'Heterocrania' which is unilateral, associated with nausea/vomiting and photo/phono phobia, and determines a timespan of several hours to a day

Galen: Introduced term 'Hemicrania'

Hippocrates: Describes unilateral visual aura and the commencing of a unilateral headache (in same half of aura) after end of aura

Posthumous publication of 'Practice of Physick' from Thomas Willis; Hypothized that vasodilation causes headache of migraine

Latham: Hypothized that visual aura is caused by cerebral arterial contraction'

Publication of 'On Megrim, Sick-Headache and Some Allied Disorders' from Levine, puts migraine on continuum of paroxysmal disorders

A. Tzanc: Ergotamine tatrate is effective for treating migraine

Wolff: Amyl Nitrite alleviates aura in doses that cause vasodilation, identifies the dura and extracerebral bloodvessels as painful structures that may be related to migraine.

±3000

±1500

± 10

±80

±170

±400

1684

1872

1873

1929

1941

BC

AD

NIH develops diagnostic criteria for migraine1962

Figure 1.1. Overview of some historical events that (partially)determine the present view of migraine.

Section 1

12

caused vasodilatation alleviated the aura258,380 and they identified the intracranial structures

(dura and extracerebral bloodvessels) that may be involved in migrainepathophysiology.81,354

In 1962 the Ad hoc committee on the classification of headache of the National

Insitute of Health developed diagnostic criteria for migraine and identified classical (withaura) and common forms (without aura),2 which were gradually modified to the presently

used criteria of the International Headache Society (IHS).145

Migraine present timeIHS criteria

The most common form of migraine is migraine without aura (MO) and accordingto the IHS,145 migraine without aura is described as an idiopathic, recurring headache

disorder manifesting in attacks lasting 4-72 hours. Typical characteristics of headache are

unilateral location, pulsating quality, moderate or severe intensity, aggravation by routinephysical activity, and association with nausea, photo- and phonophobia. The following

diagnostic criteria are used:

A. At least 5 attacks fulfilling B-D.B. Headache attacks lasting 4-72 hours

C. Headache has at least two of the following characteristics:

1. Unilateral location2. Pulsating quality

3. Moderate or severe intensity (inhibits or prohibits daily activities)

4. Aggravation by walking stairs or similar routine physical activityD. During headache at least one of the following:

1. Nausea and/or vomiting

2. Photophobia and phonophobiaE. At least one of the following:

1. History, physical- and neurological examinations do not suggest one of

the following disorders: Headache associated with head trauma, vascular disorders, non-vascular intercranial disorder, substances or their withdrawal, non-cephalic infection,

metabolic disorder, disorder of cranium, neck, eyes, ears, nose, sinuses, teeth, mouth or

other facial or cranial structures2. History and/or physical- and/or neurological examinations do suggest such

disorder, but it is ruled out by appropriate investigations

3. Such disorder is present, but migraine attacks do not occur for the firsttime in close temporal relation to the disorder.

The second most common form of migraine is migraine with aura (MA), which isdescribed as an idiopathic, recurring disorder, manifesting with attacks of neurological

Introduction

13

symptoms unequivocally localizable to cerebral cortex or brain stem, usually gradually

developed over 5-20 minutes and usually lasting less than 60 minutes. Headache, nauseaand/or photophobia usually follow neurological aura symptoms directly or after a free

interval of less than an hour. The headache usually lasts 4-72 hours, but may be completely

absent.145

The diagnostic criteria of the IHS are widely used nowadays in the research

community and ensure that various studies done using migraineurs are comparable to a

certain extent.

EpidemiologyUsing IHS criteria, the prevalence of migraine is 6% among men and 15-17%

among women (reviewed in418). In both groups, prevalence really starts to rise in the early

twenties and is highest around the age of fourty after which it declines again.418 The

prevalence of migraine without aura is generally 1.5 to 2 times higher than the prevalenceof migraine with aura.353,363 The median attack frequency in active migraineurs ranges from

0.4352 and 1.5416 to 2135 attacks per month and the median duration varies from 9 to 24

hours when using IHS criteria (reviewed in417). In comparison to other headaches, migraineis more disabling and has a higher intensity compared to other headaches417; 43% of

employed migraineurs suffer from work loss.352 Generally speaking, headache is a disorder

which is an enormous burden on society. Not only in view of the economic costs, but alsoconsidering the psychosocial costs.228 Up to 70 % of the interpersonal relationships are

impaired by migraine.95 It is remarkable that despite this burden, only 64 % of migraine

patients search medical attention and that most migraine sufferers take over-the-counterdrugs.95

Migraine pathophysiologyMigraine has received enhanced attention from the research community over the

past 33 years. The number of scientific medical publications in a National Library of Medicine

(NLM-) Medline database (Pubmed, http://www4.ncbi.nlm.nih.gov/PubMed/) using the term

‘migraine’ in title, abstract or keywords has increased steadily from 99 publications per yearin 1966 to 578 publications per year in 1998. The percentage of articles in comparison to

the total number of scientific medical publications in the NLM Medline database has

increased from 0.057% in 1966 to 0.133% in 1998 (figure 1.2), implying that the attentionfrom the research-community in migraine pathophysiology increased continuously over the

past 33 years.

http://www4.ncbi.nlm.nih.gov/PubMed/

Section 1

14

Despite this increasing attention, little is still known about the pathophysiological

mechanisms that underlie a migraine attack. Research has, however, advanced severaltheories concerning the pathophysiology of migraine in general, or the individual aspects of

a migraine attack. These will be discussed shortly.

Neurogenic inflammation theoryThe complex of trigeminal sensory afferents that innervate the dura mater and the

larger blood vessels of the brain is called the trigeminovascular system. Animal studies haveshown that upon stimulation of these trigeminal afferents, neuropeptides such as substance

P (SP) and calcitonin gene related peptide (CGRP) are released at the afferent terminal site,

causing neurogenic inflammation (NI) in the perivascular space of bloodvessels of themeninges.311 Part of the neurogenic inflammatory process is plasma protein extravasation

(PPE) and vasodilation. Using animal models it has been shown that the classic ergot

alkaloids,365 sumatriptan45 and also the new generation, centrally active triptans64 inhibitdural PPE which is induced by trigeminal afferent stimulation. Also, non-steroidal-anti-

inflammatory-drugs inhibit dural PPE48 and have been reported to be effective in the

treatment of migraine327,342. This argues for the relevance of NI in migraine. CGRP levels

1965 1970 1975 1980 1985 1990 1995 2000

0

50

100

150

200

250

300

350

400

450

500

550

600

Number of publications using term 'migraine'

Total number of publications (x1000)

Num

ber

of p

ublic

atio

ns

Year

Figure 1.2 Number of total scientific medical publications (filled squares, x1000) and thoseusing the term ‘migraine’ in title, abstract or keywords (open rounds) in a NLM Medlinedatabase (Pubmed, http://www4.ncbi.nlm.nih.gov/PubMed/) over time.

Introduction

15

present in plasma samples taken from the jugular vein from migraine patients are indeed

elevated during a migraine attack.128

These patients did not show an elevation of plasma SP levels128, which argues

against the occurrence of NI in migraine. Also, inflammation of the meninges has never

been detected in migraineurs. Most anti-migraine drugs not only ameliorate PPE but alsoinduce vasoconstriction. Bosentan, which blocks PPE without having vasoconstrictive effects,

was ineffective in alleviating migraine attacks when given during the headache phase,277

which implies that NI is not involved in migraine. However, it cannot be excluded that NIprecedes the headache phase of migraine. Therefore, treatment with drugs such as

Bosentan before the actual headache phase would be necessary to definitely prove the

irrelevance of NI in migraine.

The vascular theoryThe often-pulsating quality of a migraine headache implies that vasodilated vessels

induce the pain felt during a migraine headache. Most anti-migraine drugs have, besides

PPE inhibiting effects in animal models, vasoconstrictive properties. The vasoconstrictive

properties of anti-migraine drugs have been examined using animal models. This haselucidated that the arteriovenous anastomoses are the primary target of vasoconstriction by

anti-migraine drug.83,409 These shunts between the arterial blood supply to the brain and the

venous blood drainage are able to regulate the blood supply of oxygen and nutrients to thebrain. Dilation in the shunts normally causes a decrease in the arterial blood supply whereas

constriction causes an increased blood supply to the brain. As anti-migraine drugs have

been shown to constrict the shunts, it is tempting to speculate that the headache phase ofmigraine is caused by deficient arterial oxygen supply to the brain, which is restored by

shunt constriction induced by anti-migraine drugs. Studies that have examined the regional

oxygen extraction in the brain could, however, not find it altered in cortical areas thatshowed decreased cerebral blood flow.8,26 The constriction of shunts may attribute to

resolving migraine in other ways.

As mentioned earlier, the vascular theory which states that vasodilation of largeintracranial extracerebral vessels causes the headache of migraine was advanced by Willis

(1684). Latham put forward that cerebral vasoconstriction causes the aura phase.94 Olesen

and colleagues found that there is indeed a decrease of regional cerebral blood flow (rCBF)during the aura phase, supporting the local vasoconstriction theory of aura.328 They also

showed that the decrease of rCBF in the posterior hemisphere continued throughout the

headache phase,328 which was confirmed by others.8,26 This argues against the theory that achange from regional vasoconstriction to vasodilation causes the headache. Several reports

examined whether large cerebral vessels are dilated during a migraine attack, and although

some were able to show this for the middle cerebral artery at the headache site,106 theseresults remain controversial.86,504-506 If extracerebral bloodvessel dilation is the cause of the

Section 1

16

headache in migraine, then the trigeminal nociceptive afferents that innervate these vessels

in the dura and subarachnoid space are the most likely candidate for processing thenociceptive information to the brain, where pain sensation is experienced.

Cortical spreading depression theoryThe neurological symptoms and the reported local decrease of rCBF in cortical

areas during the aura phase may be caused by cortical spreading depression (CSD, a short-

lasting depolarization wave that moves across the cortex with a brief phase of neuronalexcitation that is immediately followed by prolonged nerve cell depression and a reduction in

regional cerebral bloodflow).207,223,224,479 It has been shown in animals that CSD is able to

activate the trigeminovascular system312 but these findings are criticized.164 Whereas thereis little doubt that vasoconstriction occurs in cerebral cortex during aura, the actual

prolonged decrease of neuronal activity has not been shown. Also, whereas CSD can be

easily initiated in animals experimentally, similar actions have failed to elicit CSD in humansubjects.122 The relevance of CSD in migraine pathophysiology, therefore, still needs to be

determined.

Deficient habituation theory of migraineExtensive research from Schoenen and colleagues have shown that migraineurs

(outside of the attack) show deficient cortical information processing (lack of habituation,hypo/hyperexcitability) to repetitive stimulation with a variety of sensory stimuli.210,379,473,474

The lack of habituation to repeated sensory information may underlie the reason that

migraineurs develop migraine from stimuli such as flickering lights and warm or crowdedplaces. Schoenen argues that deficient processing of sensory information, will lead to a

disruption of metabolic homeostasis, which through biochemical shifts will eventually result

in stimulation of the trigeminovascular system.379

Genetic theory of migraineParticularly the past few years, a completely new field of research in migraine

pathophysiology was opened by the discovery of altered genes in a special, dominant

hereditary form of migraine: familial hemiplegic migraine (FHM). The gene that codes for

the calcium P/Q type channel was found to be altered in persons suffering from FHM byOphoff and colleagues330,331,439 and later, this group also reported of evidence that the same

gene is involved in migraine with (MA) and without aura (MO).438

Other genes may also be involved in the more common forms of migraine. Theallelic distribution of the human serotonin transporter gene was found to be altered in MO

and MA compared to controls and was altered in-between patients with MO or MA as well.325

Also, a subgroup of MO patients that show dopaminergic hypersensitivity has different allelicdistribution at the locus of the dopamine D2 receptor.82 Different dopamine D2 receptor

Introduction

17

allelic distribution has also been shown in MA patients that have anxiety disorders and/or

major depression.340 Finally, the increased prevalence of migraine in females may be relatedto a ‘migraine susceptibility locus’ on the X chromosome.323

How these altered allelic distributions in various groups of migraineurs are exactly

involved in migraine pathophysiology is still speculative but they do suggest that multiplepathophysiological mechanisms may lead to one similar kind of disorder, migraine, whether

or not this is associated with hemiplegia, dopamine hypersensitivity, aura or anxiety /

depression.

Nitric oxide theory of migraineNitric oxide (NO) is a gas that easily diffuses into tissue and has acute vasodilatory

properties. The role of NO in migraine is examined predominantly by the group of Olesen,

Thomsen, Iversen and Lassen from the Department of Neurology, Glostrup Hospital

Copenhagen. Several arguments exist for relating NO to migraine pathophysiology. Thesehave been reviewed442 and will be discussed shortly.

First of all, the NO donor nitroglycerin can induce a migraine attack in migraineurs

and headache in non-migraineurs.167,169,329,442 Second, histamine can trigger migraine inmigraineurs through NO-dependent mechanisms.221 Third, nitroglycerin-induced headache

can be antagonized by the anti-migraine drug sumatriptan.168 Fourth, NO may cause the

release of CGRP from perivascular nerve endings477 a neuropeptide that is found elevated inthe jugular vein of migraineurs.128 Fifth, the vascular reactivity to NO in migraineurs is

enhanced as the dilation of the middle cerebral artery caused by NO is increased in patients

suffering from migraine441 and finally, the NOS inhibitor 546C88 has been testedsuccessfully in migraineurs.217,218

All these findings, and more,442 imply an import role of NO in migraine

pathophysiology, at least as one of the key mediators.

Cerebral theoryMigraineurs may suffer from premonitory symptoms (fatique, yawning, hungry,

higher irritability, shivering), which are different from aura symptoms, up to 48 hours prior

to the actual attack. The number of migraineurs that suffer from such pro-dromal symptoms

varies from 14%353 to 88%468 but this implies that the actual start of a migraine attack (atleast in some migraineurs) is long before the start of the aura or headache phase. The

nature of these symptoms implies that the brain itself is involved. The hypothalamus is

involved in the control of yawning,14 hunger436,443 and shivering,501 implicating theinvolvement of the hypothalamus early in the migraine attack.

Other cerebral areas that may be involved in initiating migraine are the locus

coeruleus (LC) and dorsal raphe (DR). Weiller and colleagues examined the cerebral activitypatterns of humans suffering from a spontaneous migraine attack using regional cerebral

Section 1

18

bloodflow (rCBF) measurement with positron emission tomography (PET).478 The study

showed that several cortical areas and brainstem regions were activated during a migraineattack. The activation of certain regions in the brainstem, that coincide with the location of

the DR and LC, persisted after abolition of the migraine attack with sumatriptan. This finding

led the authors to conclude that these regions may be involved in the initiation of themigraine attack.478 The LC has been noted to play a role in migraine before. Lance and

colleagues argued that the LC, due to its control on both cerebral circulation and pain

transmission at the level of spinal and trigeminal medulla, may play an essential role inmigraine. Enhanced activity of the LC may cause vascular changes in migraine, followed by

decreased activity of the LC causing attenuated inhibition on pain transmission at the level

of the spinal/trigeminal medulla.215

SummarySome theories for the pathophysiology of (aspects of) migraine have been put

forward. They do not exclude each other. It is possible, based on the various forms of

migraine and the diverse ways it manifests itself in migraineurs, that different

pathophysiological mechanisms underlie migraine. The various theories generally agreeabout one thing: the trigeminovascular system becomes activated during the most disabling

phase of a migraine attack: the headache phase.103,125,221,311,312,373,379 Many animal models

of the headache phase of migraine are therefore based on stimulation of thetrigeminovascular system.

Introduction

19

Animal models of migraine headacheThere is no animal model of migraine. We do not know whether animals do

experience migraine, but most likely, they do not. At best, animal models mimic aspects of a

migraine attack. As the term ‘model’ implies, modelling an aspect of migrainepathophysiology in animals, implies that one has to acknowledge that it only mimics the

situation in human migraineurs. A model, however, has the advantage that complex

mechanisms that underlie a migraine attack can be studied in controlled conditions. Mostanimal models published thus far have modelled the headache phase of migraine, not only

because this is the most disabling feature of a migraine attack but also because there is

little doubt that the trigeminovascular system is involved.

Animal models of trigeminovascular stimulationThe trigeminovascular system consists of the intracranial, but extracerebral

vasculature in the dura mater and the subarachnoid space that are innervated by afferents

of the trigeminal system. Anatomical studies have shown that the meningeal vasculature is

innervated by small unmyelinated sensory fibers which originate in the trigeminalganglion.238,280,281,452,454 Animal models of trigeminovascular stimulation are based on

electrical, mechanical or chemical stimulation of the trigeminovascular system. Upon

activation, trigeminal afferents transmit impulses orthodromically to synaptic nerve endingswithin layer I and II of the trigeminal nucleus caudalis (TNC I,II).286 This is the primary relay

IntermezzoFos as a marker of neural activity after nociception in conscious animals

Fos is the protein product of the proto-oncogene c-fos. It can regulate the expression of othergenes in cells. To do this, it has to form a dimer with a protein member of the Jun family afterwhich the dimmer complex can bind to the activator-protein-1 (AP-1) site in DNA gene expressionpromotor regions. The transcription of genes with an AP-1 site (for example the neuropeptidesenkephalin and substance P) can be modulated by Fos-Jun dimers.407

The value of Fos as an anatomical marker of neuronal activity after several types of stimuli,including nociception, has been discussed extensively elsewhere.43,144,304,305 The general consensusis that the presence of Fos in neurons following a painful stimulus does reflect enhanced neuronalactivity,43,144,162 but that the absence of Fos in neurons does not necessarily mean that neuronswere not activated. There are few areas in the brain that do not express Fos after painfulstimulation of which activation could be expected based on electrophysiological andneuroanatomical studies.43,144 The use of Fos as marker of neural activity has the great advantagethat it can be analysed a few hours after the experiments (expression peaks approximately 2 hrs.after stimulation), so no invasive techniques have to be used during the experiments. This allowsthe study of neural activity in conscious, unrestrained animals.

Interpretation of Fos expression results in brain sections obtained from conscious animals demandscarefully controlled experiments that enable linkage of cerebral Fos patterns to the stimulus.Neural activation revealed by Fos may relate directly to the nociceptive stimulus but may alsorelate to the behavioural and physiological adaptations induced by the nociceptive stimulus.

Section 1

20

station of the brain for nociceptive information of the trigeminovascular system. From the

TNC I,II, the signal is transduced to the cortical areas where the pain is sensed. After

nociceptive stimulation of trigeminal afferents, not only orthodromic conduction occurs butalso antidromic conduction. This will lead to the release of neuropeptides, such as SP and

CGRP at the perivascular trigeminal nerve terminals. These neuropeptides will cause NI and

PPE. It has to be noted that this antidromic conduction is not a mechanism specific for thetrigeminal system but a more general primary defence mechanism of sensory nerves against

possible tissue damage.

In the mid and late eighties, a variety of animal models have been introducedusing trigeminovascular stimulation to mimic various types of headache. Basically, they can

be characterised by 1) the type of stimulation (e.g. chemical, electrical or mechanical), 2)

the place of stimulation (e.g. trigeminal nerve, trigeminal ganglion or trigeminal afferentterminals) and 3) the markers used to measure activity in the trigeminovascular system

(figure 1.3). The latter can be divided in parameters that assess orthodromic or antidromic

activity in the trigeminovascular system. Most frequently used are Fos expression / electricalrecordings in the TNC and PPE in the dura mater respectively. Electrical stimulation of the

trigeminal ganglion combined with the measurement of PPE in the dura mater is the most

extensively used paradigm. Electrical stimulation of the trigeminal ganglion and nerve, as

IntermezzoCapsaicin in the trigeminovascular system

Capsaicin was isolated as the pungent ingredient of hot chilli peppers more than a century ago (444

cited in430). In 1967, an international journal reported that capsaicin not only activated sensoryfibers but could also block them at sufficiently high doses.173 The primary target of action ofcapsaicin are the rat unmyelinated C-polymodal nociceptors (mechanoheat, chemonociceptive,warmth) and thinly myelinated A-delta afferents (mechanoheat).429,430 This subpopulation ofcapsaicin sensitive primary afferent neurons (CSPANs) has the capability to releaseneurotransmitters from both their central and peripheral nerve endings enabling dual afferentorthodromic and efferent antidromic conduction.156,250 The neurotransmitters that are frequentlyassociated with CSPANs are the neuropeptides SP and CGRP156,250,251 but other transmitters such assomatostatin, glutamate, aspartate, vasoactive intestinal polypeptide and adenosine have also beenrelated to CSPANs (reviewed in249).

The result of antidromically CSPAN activation is an increase in vascular permeability, plasma proteinextravasation (PPE), vasodilatation and the formation of oedema, together called neurogenicinflammation (NI). SP is involved in mediating PPE38,52,56,170,194,203,371,490 whereas CGRP is a potentdilator substance27,37,39,110,115,336 and potentiates the PPE caused by SP.115 Intracranially, capsaicininduces dilatation of cerebral vessels through the release of CGRP.174

CGRP is increased in the blood of migraineurs during the attack128 pleading for the use of capsaicinin animal models that should mimic migraine-like headache. Many anti-migraine drugs reduce duralPPE in animal models,45,46,48,64,201,227,266,274,275,365,385,386,485 a process mediated by CSPANs. Also, anti-migraine drugs were effective in reducing the activity in the TNC I,II caused by intracranial afferentstimulation with capsaicin. These observations imply the occurrence of NI, and involvement ofCSPANs in migraine and the use of capsaicin as a stimulating ‘noxious’ substance in animal modelsof trigeminovascular headache.

Introduction

21

model for the vascular changes during migraine was initiated in 1984 by Lambert and

colleagues.209 The measurement of dural PPE was coupled to trigeminal ganglion stimulationa few years later by Markowitz and colleagues.261 Up until now, besides vascular

effects96,123,126,132,337,385 and dural PPE,23,24,40,46,64,119,178,227,266,283,337,381,385,410,470,497 Fos

expression in the TNC,61,201,292,344 c-Fos mRNA in the TNC,386 electrical recordings in theTNC211,420 and the neuropeptide release in the jugular vein125,127 have been used to study

the anti and orthodromic activity in the trigeminovascular system after electrical trigeminal

ganglion stimulation. Many anti-migraine drugs have been tested in these models andreduced the activity in the trigeminal system after electrical trigeminal ganglion/nerve

stimulation,45,46,48,64,201,227,266,274,275,365,385,386,485 which implicates that it is a valuable model

for the pathophysiological mechanisms that occur during the headache phase of migraine.The sagittal sinus is one of the larger blood vessels in the dura mater, innervated

by trigeminal nerves, and electrical and mechanical stimulation of this vessel has been used

to mimic migraineous headache, especially by the group of Lambert, Goadsby, Zagami andothers.132,211,499,500 The majority of these experiments were performed in cats and TNC I,II

Fos expression and TNC electrical recordings were used to assess the orthodromic

conduction characteristics of trigeminovascular afferents. Also using these models, anti-migraine drugs effectively inhibited trigeminovascular nociception.129,130,157,158,185,186,419

The dura mater is innervated by trigeminal afferents and electrical and mechanical

stimulation of the dura has therefore been used by some to activate the trigeminovascularsystem.44,73,183,291,422,484,485,488

The final type of trigeminovascular stimulation employs noxious chemical

compounds. Inflammatory soup has been applied to the dura mater,44,488 bradykinin hasbeen applied on extracerebral vessels,202 nitroglycerin was injected systemically433,434 and

blood, carrageenin321,322 and capsaicin have all been used intracisternally.75,78,79,296,297

Intracisternal capsaicin infusions, to stimulate intracranial nociceptive fibers, wasstarted in 1981.172 Intracisternal application of irritants as model of trigeminovascular

nociception was initiated by the group of Moskowitz and co-workers, who started with the

intracisternal application of autologous blood and carrageenin321,322 in 1992 but later theyswitched to capsaicin application.75,78,79,296,297 In the majority of experiment, Fos expression

in the TNC I,II was determined in order to assess activity of the trigeminovascular system.

Anti-migraine drugs also were studied successfully in this model.77,297,322

All described models of trigeminovascular stimulation, except for the experiments

of Tassorelli and colleagues,433,434 are conducted on anaesthetized animals. Anaesthesia has

the advantage, besides ethical considerations, that the reproducibility of the experimentaldesign is high. Whereas anaesthetics prevent pain sensation and the study of cerebral

processing of the pain signal, they most likely do not hinder the nociceptive processes that

generate the pain signal in the meninges or the effects that are antidromically mediated.Orthodromic conduction of the trigeminovascular system, however, as often

Section 1

22

measured by the activity of neurons in the TNC I,II, may be affected by anaesthetics as

they block the signal somewhere between the TNC and the cortex. It is not surprising

therefore that activity downstream from the TNC has been measured only sporadically in

trigeminovascular animal models.434,500 To our knowledge, behavioural responses initiated

by trigeminovascular activation have never been investigated nor quantified in animals.Availability of a validated conscious animal model of trigeminovascular activation, may not

only enable us to study the cerebral and behavioural activity associated with

trigeminovascular headache, but may be especially relevant for the treatment ofmigraineous headache, as more and more attention is paid to the development of anti-

migraine drugs with a central site of action.

We chose to modify an existing model of chemical trigeminovascular stimulation inanaesthetized animals so it could be applied in conscious animals. Anti-migraine drugs like

ergot alkaloids, sumatriptan, and NSAIDs were already tested in anaesthetized models of

Figure 1.3 Schematic representation of the various animal models of trigeminovascularactivation. Stimulation (1) of various parts of the trigeminovascular system (2) causesorthodromic and antidromic conduction to the trigeminal nucleus caudalis (TNC) andthe perivascular afferent terminal respectively (3). As result of antidromic activation SP(substance P) and CGRP (calcitonin gene related peptide) are released at the afferentterminal. To assess the orthodromic and antidromic activity of trigeminovascularafferents, various parameters are measured (4)

ElectricalChemical

Mechanical

→ Plasma protein extravasation→ Neuropeptides in jugular vein→ Vascular alterations→ CGRP-ir afferent quality in duramater

→ Fos expression→ c-Fos mRNAexpression→ Glucose utilization→ Electricalrecordings→ CGRP-ir afferentquality→ SP release

CGRP

Antidromicconduction

Orthodromicconduction

Pain sensation

TNC

BLOODVESSEL INMENINGES

Trigeminal

Afferent terminalsGanglion

Nerve

SP1

3

3

2

4

4

Introduction

23

trigeminovascular nociception.77,158,185,186,297,322 Therefore, potential anti-migraine drugs with

a possible central site of action were studied in this thesis. Also, the relationship betweenthe immunesystem and trigeminovascular nociception will be examined.

Aim and outline of this thesis.Aim of this thesis is to study physiological and pharmacological modulation of

trigeminovascular headache in a modified animal model of trigeminovascular stimulation inthe unrestrained rat.

Section 2, titled: Characterization of an animal model of trigeminovascular headache in theconscious rat, identifies the behavioural and cerebral Fos patterns associated with various

doses of intracisternally applied capsaicin in the conscious rat (see also preface on page 26)

Section 3, titled: Immunesystem modulation of trigeminovascular headache, reviews the

literature on immunesytem dysfunction in migraine and studies the modulation of

trigeminovascular nociception by infections (see also preface on page 54)

Section 4, titled: Central pharmacological modulation of trigeminovascular headache,describes the modulation of trigeminovascular stimulation by the somatostatin analogueoctreotide and the neuronal NOS inhibitor 7-NitroIndazole (see preface on page 94)

Section 1

24

Section 2

Characterization of an animalmodel of trigeminovascular

headache in the unrestrained rat

Section 2

26

PREFACE

This section contains two chapters that characterize behavioural responses and cerebral Fos

expression patterns in a modified animal model of trigeminovascular nociception in theconscious rat. By introducing a permanent cisterna magna (CM) cannula capsaicin could be

infused intracranially in the unrestrained rat (see figure 2.1). The CM is located caudal from

the fourth ventricle and contains relative large amounts of cerebrospinal fluid. Differentdoses of capsaicin were infused into the CM to study the behavioural effects of various

intensities of trigeminovascular activation and cerebral Fos expression was quantified to

identify the pattern of cerebral activation associated with trigeminovascular nociception.Chapter 2.1 concentrates on the behavioural results and the Fos expression in the TNC I,II

whereas chapter 2.2 pays attention to the cerebral Fos expression patterns.

skull dura mater

cannula

capsaicin

rostral caudal

cisterna magna

dental cement

cortex

thalamus

brain stem

cerebellum

spinal cord

Figure 2.1 Schematic representation showing the position of the cisterna magna cannula behind thecerebellum.

Trigeminovascular stimulation in conscious rats

27

Chapter 2.1

Trigeminovascular stimulation in conscious rats1

SummaryIntracisternal infusion of capsaicin was used to induce intracranial

trigeminovascular stimulation in conscious rats. Both behaviour and trigeminal nucleus

caudalis Fos expression were examined. Exploratory behaviour was dose-dependentlyreduced and different types of behaviours were induced with various doses of capsaicin.

Head grooming and scratching show that intracranial activation of trigeminal afferents can

be referred as extracranial trigeminal stimulation. Analysis of behaviour exhibited duringtrigeminovascular stimulation may provide a powerful tool to study effects of central acting

anti-migraine drugs.

1 with: W.J. Meijler and G.J. Ter Horst. Published in Neuroreport, 8 (1997) 1123-1126.

Chapter 2.1

28

IntroductionMigraine affects about 6% of the male and 15 to 17% of the female human

population.418 The pathophysiology of migraine is unclear but involvement of the

trigeminovascular system is generally accepted.125 Animal models have been developedwhich use direct electrical,386 mechanical184 or chemical321,322 stimulation of the trigeminal

nerves or ganglion to mimic vascular head pain. Originally, this concept was based on

classic studies354 showing that stimulation of dural or pial blood vessels cause head pain inhuman subjects. Anatomical studies have shown that the meningeal vasculature is

innervated by small unmyelinated sensory fibers which originate in the trigeminal

ganglion.281,452 Upon activation, these fibers transmit impulses to synaptic nerve endingswithin the Trigeminal Nucleus Caudalis (TNC).235

Expression of the protein of the immediate early gene c-fos (Fos) is thought to

reflect functional activity in neurons162,287,346 and expression of Fos in layer I and II0 of theTNC (TNC I,II0) is used to study the activity of the sensory part of the trigeminal

system.187,321,386 Anti-migraine drugs are tested in animal models that use infusion of

chemical irritants (blood, carageenan, capsaicin) into the cerebrospinal fluid (CSF) ofanaesthetized rats and guinea-pigs, after which an increase in the number of Fos positive

cells is found in the TNC I,II0. Expression can be attenuated by trigeminal nerve

transsection, destruction of small unmyelinated fibers321 and pharmacological agents thatare prescribed for the treatment of migraine, including sumatriptan, dihydroergotamine322

and valproate.75 To enable the analysis of behaviour it is of interest to develop an animal

model that uses trigeminovascular stimulation in conscious rats. Effects of analgesic drugsthat act upon central sites downstream from the TNC can be studied using behaviour

analysis. This is important, because of the recent attempts to develop anti-migraine drugs

that act upon the central components of the trigeminovascular system.440 The present studywas conducted in conscious rats to identify characteristic behavioural responses and TNC

Fos expression induced by intracisternally applied capsaicin.


29

Materials and Methods

Experiments were approved by the committee on Animal Bio-Ethics of the University of

Groningen (FDC1051). Male Wistar rats weighing 275 to 325 gr. were used. All rats were

group housed (3 rats/cage) on a light/dark regime (L/D: 08:00 h / 20:00 h). After surgeryrats were singly housed for 3 days until the start of the experiments. Food and water were

provided ad libitum.

Cisterna Magna (CM) cannulation: Surgery was conducted under semi-sterile conditions.

Rats were anaesthetized with hypnorm (0.4 ml/kg i.m.) and sodium-pentobarbital (24

mg/kg i.p.). The CM cannula was prepared from a 23G needle (0.6x25 mm, Braun,Melsungen, Germany) of which 6.5 mm was inserted into the brain. After preparing the skull

an opening (d. 1.2 mm) was drilled at the midline of the external occipital crest. The

cannula was inserted into the CM guiding it along the occipital bone. The cannula wasattached to the skull with dental cement (Kemdent, Purton Swindon, UK) and sealed with a

polyethyleen cap.

Drugs: Capsaicin (3.05 mg) was dissolved in 1 ml of saline-ethanol-Tween80 (8:1:1) and

sonicated for 5 minutes. The capsaicin infusion solution was further diluted 1:10, 1:100 and

1:1000 in saline with 0.2% Evan's Blue (Eb; Merck, Darmstadt) to yield 1000, 100 and 10nM concentrations respectively. Eb was added to determine the extent of infusion

afterwards.

Experimental procedures: Rats were placed into the observation cage (30:30:30 cm) and100 µl capsaicin (10, 100 or 1000 nM) or vehicle was infused via the CM cannula using a

microinjection pump (CMA100, Carnegie Medicin, Stockholm, Sweden ) over 2 minutes.Behaviour was recorded on videotape. Video tapes of behaviour exhibited during the 2 min.

of infusion were analysed. Behaviours scored were exploratory behaviour, head grooming,

head scratching, immobilization and escape behaviour (rapid moving around the cage withsudden turns).

Perfusion and immunocytochemistry: Two hours following infusion the rats were deeplyanaesthetized with pentobarbital and perfused (saline 1 min followed by 4%

paraformaldehyde (PF) in 0.1 M phosphate-buffered saline (pH 7.4) for 20 min). Brains were

removed and post-fixed in 4% PF. Eb staining of the brain was noted to determine theintrameningeal distribution of the infusate. Brain stem and spinal cord were cryoprotectedby overnight storage in 30% sucrose in 0,1 M phosphate buffer (pH 7.4), cut to 40-µm thick

serial coronal sections at -15°C using a cryostat microtome, and collected in 0.2 Mpotassiumphosphate-buffered saline (KPBS, pH 7.4) with sodium azide (0,1%). Free floating

Chapter 2.1

30

sections (one out of five) were immunohistochemically stained for Fos according to thefollowing protocol. After pre-incubation with normal sera (NS) and 0.3% H2O2, sections were

incubated in 2% bovine serum albumin (BSA), 2% NS and primary antibody (1:2000; CRB,

Northwich) in KPBS with 0,5% triton X-100 (KPBS-T) overnight at room temperature.Subsequently, they were incubated in 2% BSA, 2% NS and secondary antibody (1:800;rabbit α sheep IgG; Pierce, Rockford) in KPBS-T at room temperature for 2 hours. Hereafter

sections were incubated with the avidine-biotine-peroxidase complex (Vector Labs,Burlingame) in KPBS-T with 2% BSA for 2 h. at room temperature. The Ni-enhanced 3,3'-

diaminobenzidine tetrahydrochloride reaction was used to visualise the presence of

peroxidase. Intermittent washing was done with KPBS. Sections were mounted, dehydratedin graded ethanol's and xylene and coverslipped with DEPEX.

Quantification: Fos immunoreactive cells were counted at levels 1, 2, 3, 4, 5 and 6 mmcaudal from the obex by an observer blinded for the experimental procedures. For each

level up to 5 sections were counted and averaged. As there were no significant differences

in the number of c-fos positive cells between either side of the TNC I,II0, the total numberof cells per section was counted. The mean of the total TNC I,II0 was calculated by

averaging the 6 levels.

Statistics: Data were analysed using One Way Anova with Dunnett's t-test as multiple

comparison method. p values < 0.05 were considered significant. Data are expressed as

mean ± S.E.M.


31

Results

Inclusion criteria: Twenty-five rats

were included in this study. All rats in

which it was possible to extract CSFwere included. The blue staining

pattern from the Eb that was dissolved

in the infusion solution of these ratswas identical. Blue staining was

observed in the dura mater ventral

from the cerebellum, around thebrainstem and the first levels of the

spinal cord. Also, rats were included in

which infusion succeeded and thatshowed the same Eb staining pattern.

Rats with a different staining pattern

(left/right differences, no staining ofdura mater or staining of the wound

around the cannula) were excluded.

All rats had returned to pre-operativeweight by the day of the experiment.

Fos: After infusion of the variouscapsaicin concentrations into the CM,

the number of Fos positive cells at all

levels of the TNC I,II0 increased dose-dependently (table 2.1.1). The 1000

nM concentration capsaicin resulted in

a significantly increase of the averagenumber of Fos immunoreactive cells

(772 ± 52 vs. control 10 ± 2). Smaller

non-significant changes were found inthe 100 nM capsaicin group (55 ± 19).

Fos immunoreactivity was present

bilateral, with no significant left-rightdifferences and only at 1 mm caudal

from obex a slightly higher

concentration of Fos positive cells wasfound in dorsal and ventral parts of TNC I,II0. At other levels there was an equal distribution

* p< 0.05 from control. # p< 0.05 from capsaicin10 nM @ p< 0.05 from capsaicin 100 nM (ANOVAwith Dunnett's multiple comparison method).

Table 2.1.1: Nr of Fos positive cells in all treatedgroups in the TNC I,II0.

mm caudal Control Cap 10 nM Cap 100 nM Cap 1000 nM

from obex

1 7.0 ± 1.4 9.1 ± 3.6 21.0 ± 10.7 854.8 ± 37.7*#@

2 4.6 ± 1.1 6.5 ± 1.9 33.8 ± 11.4 795.1 ± 36.8*#@

3 8.5 ± 2.5 13.1 ± 3.5 48.0 ± 17.3 813.0 ± 66.8*#@

4 10.5 ± 3.5 14.8 ± 3.4 65.8 ± 23.6 847.7 ± 56.0*#@

5 13.8 ± 3.0 12.3 ± 1.7 72.5 ± 25.9 793.9 ± 115.6*#@

6 17.1 ± 5.2 12.3 ± 2.1 87.8 ± 32.8 525.7 ± 60.9*#@

Mean 10.3 ± 2.1 11.3 ± 1.9 54.8 ± 18.6 771.7 ± 51.9*#@

Figure 2.1.1 A,B: Photomicrographs of Fospositive cells in the trigeminal nucleus caudalislayer I,II0 in a control rat (A) and rats treatedwith 1000 nM Capsaicin (B). Bar = 0.2 mm

Chapter 2.1

32

of Fos positive cells throughout the TNC I,II0 (figure 2.1.1).

Behaviour: Capsaicin infusion into the CM induces different types of behaviour (Fig. 2.1.2)

during the 2 minutes of infusion. Control animals almost exclusively explore the cage (112.4± 4.5 of the 120 s): capsaicin dose-dependently reduced this exploring behaviour. Whereas

there is a strong tendency towards significance for immobilization behaviour in the capsaicin

10 nM group (24.5 ± 5.9 vs control 2.8 ± 2.6 s), only exploratory behaviour in the secondminute was significantly reduced (36.0 ± 5.1 vs control 52.6 ± 4.5 s). In the capsaicin 100

nM treated group, exploratory behaviour is reduced and there is a significant increase in

immobilization behaviour (28.4 ± 6.8 vs 2.8 ± 2.6 s). In the control group there is no headgrooming and escape behaviour observed while in the capsaicin 1000 nM these behaviours

are significantly induced (20.8 ± 2.1 and 21.8 ± 6.9 s, respectively) predominantly during

the second minute of infusion.

Figure 2.1.2: Types of behaviours observed during 2minutes vehicle infusion or various concentrations ofcapsaicin into the Cisterna Magna of unanaesthetizedrats. Vehicle treated animals (n=5). Capsaicin 10nM treated animals (n=6). Capsaicin 100 nM treatedanimals (n=10). Capsaicin 1000 nM (n=4). * p<0.05 from control. # p< 0.05 from capsaicin 10 nM @p< 0.05 from capsaicin 100 nM (ANOVA with Dunnett'smultiple comparison method).

Exploring Head Grooming Head Scratching Escape Immobilisation0

20

40

60

80

100

120

*@

@#

#

# @

Type of behaviour

*

*

*

*

Tim

e (s

ec)


33

Discussion

Ethical aspects: Although the escape behaviour in the 1000 nM group shows that the

animals were severely affected by capsaicin stimulation, this behaviour stops immediately

after infusion and immobilization behaviour was the only abnormal behaviour observed after15 min. Perfusion of the animals 2 h after initial stimulus ensured that the pain was of short

duration; however, according to the ethical guidelines for investigations of experimental

pain in conscious animals,503 the number of animals was kept as low as possible.

Evan's blue staining pattern: Eb was used to mark the intrameningeal distribution of

capsaicin. The staining of dura mater ventral from cerebellum, around brainstem and spinalcord indicates that C-fibers innervating these regions are stimulated. However, because

somatotopy of the trigeminal system has been shown regarding facial trigeminal

innervation423 and the spatial distribution of Fos immunoreactive cells after 1000 nMcapsaicin instillation was remarkably similar throughout the whole TNC I,II0, it is more likely

that trigeminal fibers around blood vessels throughout the whole subarachnoid space and

dura mater are stimulated. The difference in molecular weight and the ability of Eb to bindto proteins might explain the possible discrepancy between Eb staining and the area

stimulated by capsaicin.

Behavioural characteristics of trigeminovascular activity: The approximately 10 fold increase

in the number of Fos immunoreactive cells after an increase in capsaicin concentration from

100 to 1000 nM indicates a direct, specific, dose-dependent relationship between Fosexpression in TNC I,II0 and the capsaicin concentration used. As capsaicin selectively

activates nociceptive fibers and the principal nociceptive innervation of blood vessels of the

subarachnoid space and dura mater originates in the trigeminal ganglion, thetrigeminovascular system is slightly activated in the 100 nM capsaicin group (Fos data do

not reach statistical significance) and highly activated by 1000 nM capsaicin treated animals.

Behavioural analysis, however, showed significant changes in the 100 nM capsaicin-treatedanimals and not only showed a dose-dependent decrease in exploratory behaviour but also

a dose-dependent induction of different forms of behaviour. Immobilization behaviour was

induced in the 100 nM treated animals, whereas active behaviours such as head groomingand escape behaviour are induced in the 1000 nM capsaicin group only.

An intensively studied animal behavioural pain model is the rat formalin test.63,483

This model uses formalin injection into a hindpaw of a rat after which pain behaviour israted with weighted categories. According to this rating system immobilization is indicative

of pain and grooming is indicative of greater pain,63 confirming the results of the experiment

presented here. Although the behaviour was not scored in categories, behaviour analysisafter intracisternal applied capsaicin seems to be a valid method for evaluating pain intensity

Chapter 2.1

34

and can thus be used to study central working drugs that act to reduce trigeminal painprocessing.

Head grooming indicates that intracranial trigeminal stimulation may be referred to

topical extracranial stimulation. As intracranial and extracranial trigeminal fibers do notrepresent divergent axon collaterals that originate within the trigeminal ganglion,36 the

effect of sensitization of extracranial trigeminal fibers after intracranial trigeminal stimulation

is likely to be mediated through second-order neurons in the TNC I,II0 that receive inputfrom both extracranial and intracranial fibers.

ConclusionsBehaviour analysis combined with TNC Fos expression provides a useful model to

study central processing of trigeminal afferent stimulation. Intracranial trigeminal afferent

stimulation can be referred to extracranial trigeminal afferent stimulation.

Cerebral activity patterns

35

Chapter 2.2

Patterns of cerebral activation associated with headache in the conscious rat;a Fos-immunoreactivity study1

SummaryThis report describes cerebral activity patterns after intracranial nociceptive

stimulation in the conscious rat. Intracisternal infusion of 250 and 1000 nM capsaicin wasused to stimulate nociceptive fibers of the trigeminovascular system, and cerebral Fos

expression patterns were used as marker of neuronal activity. Areas that showed

significantly increased Fos immunoreactivity after capsaicin 250 and/or 1000 nM infusioncompared to vehicle treatment were: the trigeminal nucleus caudalis (layer I and II), the

area postrema, the nucleus of the solitary tract, the parvicellular reticular nucleus, the locus

coeruleus, the parabrachial nucleus and the dorsal, median and magnus raphe nucleus. Theventrolateral periaqueductal gray, the intralaminar thalamic nuclei, the dorsomedial,

paraventricular, ventromedial and supraoptic hypothalamic nucleus were also Fos positive

after capsaicin treatment as were the centrolateral and basolateral amygdala, parts of theprimary somatosensory cortex and the granular / dysgranular insular cortex. Most areas

affected by the treatment participate in (anti-) nociception although indirect activation by

pain-associated physiological and behavioural responses can not be excluded. IncreasedFos-ir in the locus coeruleus, the dorsal raphe and the hypothalamus after intracranial

trigeminovasucular stimulation provides evidence against a pathogenetic role of these nuclei

in migraine and cluster headache respectively, as was suggested by neuroimaging studies.

1 with: M.B. Spoelstra, W.J. Meijler, J. Korf and G.J. Ter Horst

Chapter 2.2

36

Introduction

Intracranially, the nociceptive nerves of the trigeminal system are associated with

bloodvessels that reside in the meninges. This trigeminovascular system is thought to be theanatomical substrate for (neuro-)vascular headaches like migraine and cluster

headache.278,310 There is little known about what cerebral nuclei are activated during

trigeminovascular headaches. Essential data in this respect was provided by Weiller andcolleagues who examined the cerebral activity patterns of humans suffering from a

spontaneous migraine attack using regional cerebral bloodflow (rCBF) measurement with

positron emission tomography (PET).478 The study observed activation of several corticalareas and brainstem regions during a migraine attack. The activation of certain regions in

the brainstem that coincide with the location of the dorsal raphe nucleus (DR) and locus

coeruleus (LC) persisted after abolition of the migraine attack with sumatriptan, leading theauthors to conclude that these regions may be involved in the initiation of the migraine

attack. Crucial to the conclusion that these regions are specifically involved in migraine is

whether or not non-migraineous types of trigeminal nociception are also able to induceactivation of the DR and LC. Therefore, a recent study from the same group described that

subcutaneous injection of the irritant capsaicin into the forehead was not able to induce

activation of these specific regions in the brainstem279 supporting their previous data.478

However, migraine is a diffuse, badly localized, deep, intracranial pain, whereas the

experimental pain caused by subcutaneous capsaicin is superficial, sharp, well localized and

extracranial. It has been described in animal models that superficial pain and deep painelicits different activation patterns in the brain.188 Of course it is ethically and technically

difficult to induce intracranial experimental pain in humans, but in animal models this is

quite commonly performed. Chemical,296,322 electrical159,187 and mechanical421 stimulation ofintracranial trigeminal nerves is often used to mimic vascular headaches. Trigeminal

stimulation in these animal models induces activation of the TNC I,II; the primary target of

intracranial nociceptive trigeminal afferents. All studies used expression of the proto-oncogene protein Fos to assess the neuronal activity in the TNC I,II. Some studies also

described Fos expression patterns in other parts of the brainstem and spinal cord,159,296,322

but thus far, none of them described headache-induced Fos immuno-reactivity (Fos-ir) inthe rest of the brain. This is most likely because all studies used anaesthetics. Most

anaesthetics effectively block the pain signal somewhere along the line from nociceptive

afferent to the sensory cortex, rendering pain models using anaesthetics unfit to studycerebral activity patterns. Also, anaesthetics themselves induce Fos expression in the

brain432 thus hampering the tool of Fos expression as specific cerebral neuronal activity

marker after painful stimulation. Therefore, we developed an animal model of intracranialtrigeminovascular stimulation in the conscious rat.192 Intracisternal infusion of different

concentrations of the irritant capsaicin was used to activate intracranial nociceptive nerves


37

in unanaesthetized rats. Cerebral neuronal activity was assessed using Fos

immunocytochemistry. The cerebral nuclei exhibiting Fos-ir are discussed in light of theirpossible role in (anti)-nociception and in light of the cerebral patterns found by PET-scan in

migraine and cluster headache patients.

Chapter 2.2

38

Methods

AnimalsMale Wistar rats weighting 310 ± 8 gr. were used. All rats were housed group wise

(3 rats/cage) on a light/dark regime (L/D: 08:00 h / 20:00 h) and surgery was performed 5

days after arrival. After surgery rats were single housed for 3 days until the start of the

experiments. Food and water were provided ad libitum. Experiments were approved by thecommittee on Animal Bio-Ethics of the University of Groningen (FDC 1051, FDC 1191) and

performed according to the ethical guidelines for investigations of experimental pain in

conscious animals.503

Surgical proceduresCannula's, surgical materials and rat skin were disinfected with 0.5% chlorhexidine.

All rats were anaesthetized with 0.4 ml/kg i.m. hypnorm (fentanyl 0.3 mg/ml and fluanisone

10mg/ml; Janssen, Beerse, Belgium) and pentobarbital (24 mg/kg i.p.). A midline incision in

the skin at the top of the head was made and membranes from the parietal, interparietaland rostrodorsal part of the occipital skull were removed.

The cisterna magna cannula was prepared from a stainless steel needle (0.6x25

mm, 23G x 1"; Braun, Melsungen, Germany) which was shortened to 6.5 mm. Rats wereplaced in a stereotaxic apparatus with incisor bar at –7 mm from the horizontal plane. Two

holes were drilled into the caudal corners of the interparietal skull and 2 screws were driven

1.5 mm into the skull. A hole (d. 1.2 mm) was drilled at the midline of the external occipitalcrest for placement of the cisterna magna cannula. The cisterna magna cannula was

carefully placed through the hole with a horizontal rostro-caudal approach and pushed

beneath the dorsal part of the occipital bone until the dorso-caudal part of the occipital bonewas reached. Then the cannula was slowly turned from the horizontal, rostral-caudal plane

into the dorsal-ventral plane. Guiding it along the occipital bone caudal from the cerebellum,

the cannula was gently positioned into the cisterna magna. Correct placement of thecannula was confirmed by withdrawal of CSF after which the cannula was fixed to the skull

with dental cement (Kemdent, Purton Swindon, UK) and closed with a piece of silicon tube.

The wound was sutured and rats were allowed to recover for 3 days.

Experimental procedures

InfusionRats were placed into the experimental cage (30, 30, 30 cm) and capsaicin (250

nM or 1000 nM) or vehicle was infused into the cisterna magna with a microinjection pump(CMA100, Carnegie Medicin, Stockholm, Sweden). Rats received 100 µl capsaicin in 2

minutes.


39

Perfusion and immunocytochemistryRats were perfused 2 h. following infusion of capsaicin or vehicle. Prior to the

transcardial perfusion rats were deeply anaesthetized with sodium pentobarbital and

perfused with 0.9% saline for 1 min, followed by 4% paraformaldehyde (PF) in 0.1 Mphosphate-buffered saline (pH 7.4) for 20 min. After removal of the occipital bone,

placement of the cannula in the cisterna magna was confirmed and extent of the infusion

into the epidural space was determined by inspection of the Evans Blue (dissolved (0.2%) inthe capsaicin and vehicle solutions) staining. After the removal, the brains were post-fixed in

4% PF during 24 h. Prior to sectioning the brain was cryoprotected by overnight storage in30% sucrose in 0.1 M phosphate buffer (pH 7.4). Forty µm thick coronal serial sections

were prepared on a cryostat microtome at -15°C, and collected in 0.2 M

potassiumphosphate-buffered saline (KPBS, pH 7.4) with sodiumazide (0.1%).

Free floating sections were immunocytochemically stained for Fos protein accordingto the following protocol. Sections were rinsed 3x10 min. in KPBS, pre-treated with 0.3 %

H2O2 in KPBS for 10 min, rinsed 3x10 min. in KPBS and pre-incubated in 2% bovine serum

albumin (BSA; Merck, Darmstadt, Germany), 2% normal serum (NS, normal rabbit serum,Sigma Chemie, Bornem, Belgium) in KPBS for 4 h. at room temperature. Subsequently,

sections were incubated in 2% BSA, 2% NS and primary antibody sheep-anti-c-fos (1:2000;

Cambridge Research Chemicals, Northwich, UK) in KPBS with 0.5% triton X-100 (KPBS-T;Bayer, Deventer, Netherlands) overnight at room temperature. Sections were rinsed 3x10

min. in KPBS and incubated in 2% BSA, 2% NS and second antibody (1:200 biotinylatedrabbit-α-sheep IgG (Pierce, Rockford)) in KPBS-T at room temperature for 2 hours. After

3x10 min. washes in KPBS, sections were incubated in avidine-biotine-peroxidase complex

(Vector Labs, Burlingame) in KPBS-T with 2% BSA for 2 h. at room temperature. Hereafter,

sections were washed in 3x10 min. KPBS and 2x10 min. in 0.1M sodiumacetate buffer(NaAc, pH 6.0). For the final staining procedure 3.3'-diaminobenzidine tetrahydrochloride

(0.05%) and ammoniumchloride (0.04%) were dissolved in 1/2 v distilled water and 1/2 v

NAS solution (5% NikkelAmmoniumSulfate dissolved in 4/5 v 0.2M NaAc and 1/5 v distilledwater). To start the diaminobenzidine reaction 0.3% H2O2 was added. The reaction was

stopped after 20 minutes. Sections were washed 2x10 min. in 0.1M NaAc and 3x10 min. in

KPBS, mounted on gelatin coated slides, air dried, dehydrated in graded ethanol's and xyloland cover-slipped with DEPEX. All staining procedures were with gentle agitation.

QuantificationTNC layer I, II.

Fos-ir cells were counted at -1, -2, -3, -4, -5 and -6 mm caudal from obex by an

observer blinded from experimental procedures. Sections from -0.5 to -1.5 mm wereaveraged to obtain the count for the -1 mm level and so on. To obtain accurate sampling of

Chapter 2.2

40

sections for each level, the trigeminal nucleus of one rat was dissected (40 µm, freezing

microtome) from obex to -7 mm from obex and all sections were immediately mounted on

gelatin coated slides. A Nissl staining was performed to show the cytoarchitecture of the

sections and Fos stained sections were compared to these Nissl-stained sections todetermine the location of the Fos-ir cells from obex. Because there were no significant

differences in the number of Fos positive cells between the right or left side of the TNC I,II,

the total number of cells per section was counted. The mean of the total TNC I,II wascalculated by averaging the Fos expression at the 6 levels.

Cerebral Fos expressionTo delineate the cerebral areas we primarily used the atlas of Paxinos and Watson,

1997. Some details, like the laminar organization of the cortex, were delineated using the

atlas of Swanson, 1992.Fos stained sections of all animals were first scanned to qualify the areas that

showed substantial Fos expression in one or all of the three groups. These areas where

subsequently quantified by an observer blinded from the treatments. Also, areas that didn’tshow substantial Fos expression but were relevant according to the literature on pain

perception and responses were also quantified. At least 2 sections, but often more

(depending on the rostro-caudal extend of the area counted), per area were counted andaveraged to establish the number of Fos positive cells in each area for each individual

animal. Group averages were calculated from the individual means per area.

DrugsThe capsaicin stock solution (3.05 mg capsaicin per 1 ml of vehicle stock (saline-

ethanol-Tween80 (8:1:1)) was diluted 1:10 or 1:40 in saline to which 0.2% Evan's Blue(Merck, Darmstadt) was added. This yields the 1000 and 250 nM capsaicin concentration

respectively. Vehicle stock was diluted 1:10 in saline to be used as control solution.

Statistical analysesThe One Way ANOVA with Student-Newman-Keuls test as multiple comparison

method (pairwise) was used to test differences between the 3 groups. In cases of non-normal distribution or unequal variance, the Kruskall-Wallis ANOVA on ranks with Dunn’s

test as multiple comparison (pairwise) was performed. p < 0.05 was considered significant.


41

Results

In table 2.2.1, the mean numbers of Fos positive cells per (sub) nuclei is shown for

rats treated with vehicle (control, n=5), capsaicin 250 nM (C250, n=8) or capsaicin 1000 nM

(C1000, n=4).

HindbrainBoth capsaicin 250 nM and 1000 nM significantly induce Fos-ir in the TNC I,II

compared to control (103 ± 22, 772 ± 52, 10 ± 2 respectively). This difference is apparent

throughout the whole rostro-caudal extend of the TNC. Two other areas in the brainstem

show robust induction of Fos-ir after (especially 1000nM) capsaicin treatment: thecaudomedial nucleus of the solitary tract (cmNTS - Control: 4 ± 2, C250: 69 ± 38, C1000:

482 ± 94) and the area postrema (AP - Control: 4 ± 3, C250: 39 ± 23, C1000: 281 ± 96).

Although the Fos expression in the TNC layer V was generally increased in the capsaicintreated groups, the differences (in)between groups was not significant (p = 0.056). No

capsaicin-induced differences were found in layer X of the spinal cord, the trigeminal

nucleus oralis (TNO) or the trigeminal nucleus interpolaris (TNI). The only areas in thebrainstem that show significant induction of Fos after 250 nM capsaicin are the caudal

lateral NTS (control: 2 ± 1, C250: 11 ± 3), the parvicellular reticular nucleus (PCRt - control:

8 ± 3, C250: 36 ± 7), the LC (control: 5 ± 1, C250: 28 ± 8 (figure 2.2.1A)), the lateralparabrachial nucleus (lPBA - control: 21 ± 5, C250: 117 ± 22) and the medial parabrachial

nucleus (mPBA- control: 3 ± 1, C250: 23 ± 5). Except for the mPBA, these areas also show

a significant higher number of Fos positivecells in the C1000 treated animals, when

compared to C250 treated animals (caudal

lateral NTS: 22 ± 2, PCRt: 54 ± 3, LC: 71 ±4, lPBA: 274 ± 23 (figure 2.2.2)). The

median raphe nucleus and raphe magnus

nucleus (RMg) only show enhanced Fosexpression after 1000 nM capsaicin (control:

2 ± 0, C1000: 14 ± 2, control: 6 ± 2,

C1000: 31 ± 6 respectively).

Figure 2.2.2. Photomicrograph of Fosexpression in the parabrachial area of a rattreated with 1000 nM capsaicin.

Chapter 2.2

42

Figure 2.2.1. Photomicrograph of Fos expression in the locus coeruleus (A), dorsal raphe nucleus (B)and paraventricular hypothalamic nucleus (C) of a rat treated with 1000 nM capsaicin (right-side) orvehicle (left-side).


43

MidbrainMidbrain areas that showed enhanced Fos expression after (1000nM) capsaicin

treatment were the ventral (control: 33 ± 10, C1000: 95 ± 20) and the ventrolateral

(control: 89 ± 10, C1000: 197 ± 28) dorsal raphe nucleus (figure 2.2.1B) and the

ventrolateral periaqueductal gray (vlPAG - control: 100 ± 18, C1000: 257 ± 39). Otherportions of the PAG did not show significant enhancement of Fos-ir after capsaicin

treatment.

Forebrain, subcorticalAll intralaminar thalamic nuclei showed enhanced Fos expression after C250 and

C1000 treatment compared to control animals but it was significant only after 1000 nM inthe central medial thalamic nucleus (CM - control: 74 ± 14, C1000: 201 ± 51). Two other

medial thalamic nuclei, the reuniens and rhomboid thalamic nuclei, exhibit significant

increased Fos expression at 250 nM capsaicin only (control: 5 ± 1, C250: 17 ± 5; control:14 ± 5, C250: 61 ± 15 respectively) and Fos expression in nucleus submedius or in the

ventrobasal thalamic nuclei (posterior (Po), ventral posterolateral (VPL), ventral

posteromedial (VPM)) is nearly absent in all groups.Capsaicin 1000 nM induced significant increased Fos expression in the central

amygdaloid nucleus (CeA - control: 24 ± 3, C1000: 281 ± 101) and the medial amygdaloid

nucleus (MeA - control: 79 ± 16, C1000: 321 ± 59) whereas C250 induced significant Fosexpression in the CeA (C250: 137 ± 23) and the basolateral amygdaloid nucleus (BLA -

control: 26 ± 2; C250: 94 ± 19).

Most subnuclei of the hypothalamus exhibit enhanced Fos expression after 250 and1000 nM capsaicin (dorsomedial (DMH): control: 144 ± 16, C250: 245 ± 32, C1000: 331 ±

45; paraventricular (PVH, figure 2.2.1C): control: 59 ± 9, C250: 306 ± 76, C1000: 396 ±

56, supraoptic (SO): control: 9 ± 2, C250: 88 ± 24, C1000: 167 ± 26) compared to control.Fos expression in the ventromedial hypothalamic nucleus (VMH) is only enhanced after 1000

nM (control: 22 ± 3, C1000: 105 ± 39) and no significant effects of capsaicin were observed

in the lateral hypothalamic area (LH).

Forebrain, CorticalFos expression in capsaicin treated animals is generally higher in all cortical areas

studied, compared to control. Significance, however, is only reached in some layers of the

primary somatosensory cortex (SI), forelimb region (layer 5: control: 7 ± 1, C1000: 41 ±

17; layer 6: control: 40 ± 3, C250: 84 ± 13), all layers of the SI, upper lip region and thegranular and dysgranular insular cortex (control: 34 ± 6; C250: 115 ± 18; C1000: 131 ±

42). Fos expression in all other cortical areas (cingulate cortex, prelimbic cortex, agranular

insular cortex, motor cortex and SI, jaw region, oral surface) was not significantly differentbetween groups.

Chapter 2.2

44

HindbrainArea (distance to bregma in mm.) Control C250 C1000

cervical spinal cord, level 3, layer X 4 ± 1 5 ± 1 7 ± 2

level 1, layer X 4 ± 1 7 ± 1 10 ± 2spinal trigeminal nucleus, caudal part, layer I,II (-17.68<>-19.68) 14 ± 3 118 ± 25 * 722 ± 63 * #

(-14.68<>-16.68) 7 ± 2 89 ± 20 * 821 ± 42 * #

caudal, layer V (-16.68) 5 ± 1 19 ± 7 32 ± 6interpolar (-13.3) 33 ± 15 49 ± 13 111 ± 40

oral (-10.52) 4 ± 2 5 ± 2 5 ± 2area postrema (-13.68) 4 ± 3 39 ± 23 281 ± 96 * #

nucleus of the solitary tract, caudal, lateral (-13.68) 2 ± 1 11 ± 3 * 22 ± 2 * #

medial 4 ± 2 69 ± 38 482 ± 94 * #

rostral, lateral (-12.3) 2 ± 0 8 ± 2 9 ± 3medial 3 ± 1 13 ± 4 28 ± 9 * #

parvicellular reticular nucleus (-12.3) 8 ± 3 36 ± 7 * 54 ± 3 * #

raphe magnus nucleus (-11.3-10.3) 6 ± 2 13 ± 3 31 ± 6 * #

lateral parabrachial nucleus, lateral (-9.3) 21 ± 5 117 ± 22 * 274 ± 23 * #

medial 3 ± 1 23 ± 5 * 34 ± 5 *

median raphe nucleus (-8) 2 ± 0 5 ± 1 14 ± 2 * #

locus coeruleus (-9.3<>-10.3) 5 ± 1 28 ± 8 * 71 ± 4 * #

MidbrainArea (distance to bregma in mm.) Control C250 C1000

dorsal raphe nucleus, dorsal (-7.8) 7 ± 4 4 ± 1 17 ± 10

ventral 33 ± 10 32 ± 8 95 ± 20 * #

ventrolateral 89 ± 10 115 ± 22 197 ± 28 * #

periaqueductal gray, dorsolateral (-7.8) 14 ± 1 26 ± 5 36 ± 24dorsomedial 60 ± 19 74 ± 9 59 ± 21

lateral 126 ± 24 112 ± 15 147 ± 31ventrolateral 100 ± 18 135 ± 23 257 ± 39 * #

periaqueductal gray, dorsolateral (-6.8) 19 ± 3 14 ± 3 11 ± 2

dorsomedial 45 ± 8 31 ± 7 27 ± 5lateral 129 ± 24 85 ± 8 90 ± 16

Table 2.2.1. Numbers of Fos immunoreactive cells in studied areas after intracisternal infusion ofvehicle (control, n=5), capsaicin 250 nM (C250, n=8) or capsaicin 1000 nM (C1000, n=4). Dataexpressed as mean ± S.E.M.*= significantly different from control, #= significantly different fromC250.


45

Forebrain, subcorticalArea (distance to bregma in mm.) Control C250 C1000

amygdaloid nucleus, basolateral (-2.8) 26 ± 2 94 ± 19 * 44 ± 3 #

central 24 ± 3 137 ± 23 * 281 ± 101 * #

medial 79 ± 16 127 ± 18 321 ± 59 * #

thalamic nucleus, centrolateral (-2.8) 21 ± 5 41 ± 5 42 ± 5

central medial 74 ± 14 117 ± 18 201 ± 51 * #

mediodorsal 26 ± 7 67 ± 17 52 ± 13

paracentral 10 ± 3 13 ± 2 23 ± 8paraventricular 100 ± 12 251 ± 56 151 ± 24

reuniens 5 ± 1 17 ± 5 * 8 ± 1

rhomboid 14 ± 5 61 ± 15 * 19 ± 12 #

submedius 0 ± 0 2 ± 1 2 ± 1posterior thalamic nuclear group 0 ± 0 5 ± 3 1 ± 1

ventral posteromedial/lateral 2 ± 1 6 ± 2 4 ± 2

hypothalamus, dorsomedial (-2.8) 144 ± 16 245 ± 32 * 331 ± 45 *lateral 25 ± 7 40 ± 8 32 ± 8

ventromedial 22 ± 3 59 ± 12 105 ± 39 *paraventricular (-1.4<>-1.8) 59 ± 9 306 ± 76 * 396 ± 56 *

supraoptic (-1.4<>-1.8) 9 ± 2 88 ± 24 * 167 ± 26 * #

zona incerta (-2.8) 28 ± 9 46 ± 6 30 ± 7

hippocampus (-2.8) 63 ± 9 113 ± 9 * 53 ± 10 #

accumbens nucleus, core (1.6) 122 ± 24 95 ± 6 125 ± 35

shell 230 ± 10 361 ± 58 422 ± 76

Forebrain, corticalArea (distance to bregma in mm.) Control C250 C1000

motor cortex, primary (1.2) 154 ± 27 384 ± 89 240 ± 39

secondary 517 ± 60 699 ± 93 396 ± 99

primary somatosensory cortex, forelimb region, layer 1,2,3 (1.2) 26 ± 5 32 ± 6 37 ± 12

layer 4 38 ± 9 57 ± 10 56 ± 15layer 5 7 ± 1 20 ± 4 41 ± 17 *layer 6 40 ± 3 84 ± 13 * 71 ± 10

primary somatosensory cortex, jaw region, layer 1,2,3 (1.2) 75 ± 11 147 ± 27 164 ± 37

layer 4 156 ± 33 386 ± 71 310 ± 96layer 5 20 ± 4 73 ± 20 119 ± 57

layer 6 145 ± 16 411 ± 85 385 ± 88

primary somatosensory cortex, jaw region, oral surface, layer 1,2,3 (1.2) 20 ± 3 99 ± 36 64 ± 7 layer 4 15 ± 5 290 ± 122 136 ± 32

layer 5 6 ± 2 46 ± 20 32 ± 8

layer 6 38 ± 7 249 ± 82 140 ± 26

primary somatosensory cortex, upper lip region, layer 1,2,3 (1.2) 13 ± 1 61 ± 10 * 60 ± 8 *layer 4 8 ± 2 160 ± 36 * 134 ± 22 *layer 5 7 ± 2 44 ± 10 * 43 ± 8 *layer 6 31 ± 3 273 ± 57 * 174 ± 21

agranular insular cortex (1.2) 44 ± 8 79 ± 11 73 ± 32granular / dysgranular insular cortex (1.2) 34 ± 6 115 ± 18 * 131 ± 42 *

cingulate cortex, area 1 (1.2) 167 ± 12 227 ± 27 144 ± 21

area 2 94 ± 7 192 ± 29 160 ± 30cingulate cortex, area 1 (3.7) 144 ± 23 195 ± 42 247 ± 48

prelimbic cortex (3.7) 251 ± 45 287 ± 67 315 ± 55

Table 2.2.1, continued.

Chapter 2.2

46

Discussion

Intracisternal infusion of capsaicin in conscious rats induces a distinctive pattern of

Fos expression in the brain. Trigeminal pain processing pathways (TNC I,II, SI), paininhibitory nuclei (LC, DR, RMg) and many areas of the central autonomic nervous system,368

including the NTS, AP, PBA, CeA, vlPAG, PVH, DMH and CM, show enhanced Fos expression.

Notably, little Fos-ir was observed in the ventrobasal thalamic nuclei and in the agranularinsular, cingulate and prelimbic cortex there was no significant treatment effect.

The unanaesthetized setup and use of Fos expression as marker for neuronal

activity causes some limitations to the interpretation of the reported results. It is likely thatexpression of Fos is not only caused by the trigeminovascular nociceptive stimulation but,

because of the unanaesthetized conditions, also by the physiological and behavioural

responses that are elicited by intracisternal infusion of capsaicin. It can be discussedwhether these areas must be regarded as ‘false-positive’ areas, for the behavioural and

physiological responses are an intrinsic, necessary part of the pain response, enabling the

animal to cope with the painful stimulation. The areas indirectly activated by thephysiological and behavioural responses to intracranial trigeminal nociception, are hard to

identify. As we will discuss, many of the areas that showed enhanced Fos expression after

intracisternal capsaicin treatment have a potential function in (anti-) nociception. This mayvery well be the reason for the expression of Fos in these areas, as pain is a strong sensory

stimulus. To discern direct (nociceptive related) from indirect (related to the physiological

and behavioural responses induced by nociception) cerebral activation, other experimentalsetups can be used.62,140

A second limitation is caused by the use of Fos-ir as marker for neuronal activity.

Although Fos-ir can be used as marker for neuronal activation after nociceptivestimulation,43,144,162 the absence of Fos-ir in neurons doesn’t necessarily mean that these

neurons do not participate in the response.43,144 These false-negative areas may be exposed

by comparing this study to similar experiments that employ other markers of neuronalactivity. To our knowledge however, there are no studies that use intracranial, nociceptive

stimulation in conscious animals that use a different marker of neuronal activity. The

discussion will therefore focus on areas that are Fos positive.

Hindbrain

The TNC receives a dorso-ventral,388 and rostro-caudal423 somatotopic input fromall tree branches of the trigeminal nerve.471 The first, opthalmic, division projects to the

ventrolateral part of the TNC, the second, maxillary, branch projects to the mediolateral part

of the TNC and the third mandibular division terminates in the dorsomedial part of the TNC.The present finding of Fos expression in the outer layers of the TNC throughout all its


47

dorso-ventral extend, confirms the findings in rat,9 cat281,414,415 and human196,338 that all tree

branches innervate the meninges and meningeal vasculature.The largest portion of both intracranial79,158,192,193,322,421,421 and

extracranial6,12,268,288,324,425,472 nociceptive trigeminal afferents terminate in layer I and II0 of

the TNC.176,421 Other termination sites reported in various animal experiments are layer I, IIof the upper cervical dorsal horn,158,425 layer V of the TNC,6,366,421,472 layer X of the upper

cervical spinal cord187 and some parts of the TNO.12,324,349,350 Of these termination sites

mentioned in other reports, only layer I, II of the upper cervical spinal cord also showssignificant increased Fos expression after capsaicin treatment. No Fos expression was seen

in the TNO and there was no significant difference in either the Fos expression in layer V of

the TNC or layer X of the cervical spinal cord between control and capsaicin treated rats.Lack of capsaicin-induced Fos expression in these areas may be expleained by the different

kinds and locations of the stimuli used in the various experiments.

Direct trigeminal projections to the ventrolateral NTS have been reported.13,65 Ourresults confirm this, as a dose dependent enhancement of Fos positive cells is observed in

the lateral NTS at the level of the AP after capsaicin treatment. However, a more robust

increase of Fos expression is found in the medial portion of the NTS at obex level. Both themNTS and the AP receive an intense innervation from vagal nerves.65 As vagal afferents are

essential in relaying input to the central autonomic nervous system368 and the experiments

were performed in conscious rats, the Fos expression in the mNTS and AP may be derivedfrom vagal afferents that are involved in the physiological response of the rat to the pain.

The latter option is supported by a report that trigeminally mediated nociception from the

nasal mucosa induces a pressor response that is mediated by the medial NTS.91

The PCRt, the LC and the lPBA area are regions more rostrally in the hindbrain that

show a clear-cut dose dependent Fos-ir response to increasing concentrations of capsaicin.

The PCRt receives trigeminal input171 but it is unclear whether this is of nociceptive origin.Our results suggest that it is nociceptive, although the grooming and scratching of the head

that is induced by the capsaicin192,193 may indirectly elicit trigeminal activation of non-

nociceptive origin.The LC has been shown to receive direct projections from layer I of the TNC in cat

and monkey,71 suggesting that the activation in the LC is derived from activated neurons in

the outer layers of the TNC. The well-described role of the LC in pain control,259 bothdescending175,179,482 and ascending,482,502 and the close innervation of the TNC by LC

neurons108 argue that the LC is involved in a direct antinociception feedback loop activated

by the neurons in the outer layers of the TNC.The lPBA is one of the key areas in autonomic control. Switching on of this area, or

at least part of it, probably occurs directly from second order trigeminal neurons in layer I of

the TNC, as innervation of the external lateral PBA from these neurons has beendemonstrated.176 Next to this (spinal)-trigemino-nociceptive innervation, the lPBA receives

Chapter 2.2

48

input from the NTS (especially the dorsomedial portion) and the AP,152 two areas that showrobust Fos expression after capsaicin treatment in our experiments. The projection from the

NTS and AP to the parabrachial nucleus is topographically organized.152 Our results confirm

this topographical organization as the most robust Fos expression is found in the medialportion of the NTS and AP at the level of obex, combined with pronounced Fos expression in

the lateral portion of the PBA. Thus, besides receiving input from layer I of the TNC, the

lPBA area is heavily innervated by the NTS and AP. This innervation, combined withprojection patterns to forebrain areas like the amygdala, intralaminar thalamic nuclei and

the PVH (reviewed in368), which all show enhanced Fos expression after capsaicin, make it

one of the key areas to modulate forebrain regions that are involved in autonomic /emotional control.

Serotonergic neurons from the RMg project directly on nociceptive neurons in the

TNC.233,245 Stimulation of the RMg inhibits nociceptive neurons in the TNC, both in rats246

and cats.88 This pathway of anti-nociception is most likely activated in the group of animals

treated with 1000 nM capsaicin as these animals show a small but significant increase of

Fos-ir in the RMg compared to control animals.

Midbrain Nuclei,The dorsal raphe (DR), one of the nuclei shown to be activated during a migraine

attack, even after abolishment of the attack with sumatriptan478 showed enhanced Fos

expression after intracranial trigeminovascular stimulation with a high concentration of

capsaicin. The DR receives a substantial projection from the lPBA.369 As the lowerconcentration of 250 nM was not sufficient to activate the DR, it can be concluded that

robust stimulation is necessary for DR activation. The DR is well known for its involvement

in modulation of anti-nociception in several pain models90,198,236,267,426 which may be thefunctional explanation for enhanced Fos expression found in the 1000 nM capsaicin treated

animals. The serotonergic ventrolateral DR neurons project directly to the TNC.234 Together

with the projection from the DR to the LC199 (which also projects back to the TNC108) thisprovides possibilities for the DR to influence trigeminal nociception through serotonergic and

noradrenergic mechanisms.

The PAG is a complex structure with differential functionality including analgesiaand autonomic regulation.17 The PAG is involved in antinociception88,232,300,306,332 and

especially the ventrolateral PAG (vlPAG) shows enhanced Fos expression in various rat

models of deep somatic and visceral pain.62 A study from Keay and Bandler showed thatwhereas cutaneous noxious stimulation induces Fos in the lateral and dorsolateral segments

of the PAG, deep noxious stimulation induces Fos in the ventrolateral portion of the PAG.188

More recently they also showed that activation of the vlPAG is associated with intracranialnociception in a sagittal sinus stimulation model in the anaesthetized cat.189 This is

confirmed by the present finding of enhanced Fos-ir in the vlPAG after trigeminovascular


49

activation by intracisternal capsaicin in conscious animals. The results suggest that

intracranial nociceptive trigeminovascular stimulation is perceived as deep noxious pain.In cats, stimulation of the vlPAG results in inhibition of nociceptive neurons in the

TNC,88 suggesting a role in anti-nociception for this area. The vlPAG, however, also shows

increased Fos expression after for example brief social stress293 or restraint stress448 and hasin more general terms been associated with the quiescence response after a stressful

encounter.244 Therefore, it is likely that activation of the vlPAG is not specific for certain

types of pain, but more generally for certain types of stressors, namely, those that induce aquiescence response. Although the animals treated with 1000 nM capsaicin in our

experiments initially show active types of behaviour during the infusion of capsaicin, the

subsequent 10 minutes after infusion are indeed increasingly dominated by immobilization(unpublished data). The Fos-ir response in the vlPAG is probably dependent on the intensity

of the stressor as the Fos expression in the 250 nM treated animals is not different from

control animals. This dependency of vlPAG Fos-ir on the intensity of the stressor isconsistent with an animal model in which noxious colorectal distension is able to further

enhance the Fos induction caused by loose restraint.448

Forebrain Nuclei, subcortical,In subcortical forebrain, capsaicin activated amygdaloid, medial thalamic and

hypothalamic nuclei. The basolateral, medial and central nuclei of the amygdala have allbeen associated with several aspects of anti-nociception. The MeA was related to post-

stress-induced analgesia,481 the BLA to fear conditioning of a nociceptive signal387 and direct

antinociception148,334 and the CeA to antinociception pathways.254,255,334 The CeA receivesinput from the outer layers of the TNC through the lateral parabrachial area176 and from the

NTS, directly and also through the PBA.368 As the outer layers of the TNC, the caudo-medial

NTS and the lPBA show increased Fos-ir after capsaicin treatment, Fos expression in theCeA after capsaicin treatment is most likely the result of activation of these brainstem

nuclei. The input of the CeA from the NTS, lPBA and also from the agranular insular

cortex491 argue that, next to having a role in anti-nociception, the CeA has an importantgeneral integrative role in autonomic functions related to pain.

Although it is only significant in the CM, the intralaminar thalamic nuclei show

enhanced Fos expression after capsaicin treatment. The intralaminar nuclei, including theCM, receive input from the lPBA,111,154 which in turn is innervated by the outer layers of the

TNC and spinal cord.53,402 This projection pathway suggests that the intralaminar thalamic

nuclei are involved in the arousal associated with pain.154,368

None of the ventrobasal thalamic nuclei, the VPM, VPL and Po, exhibit enhanced

Fos expression after capsaicin treatment compared to control, although the VPM and Po

receive input from the TNC I,II.389,390 Especially the VPM has been shown to becomeactivated after trigeminal nociception in anaesthetized animal models using local cerebral

Chapter 2.2

50

glucose utilization134 and single cell recordings495 to assess neuronal activity. It is not likelythat the use of anaesthetics somehow induces this activation of the VPM in these models

because in an anaesthetized animal model using Fos-ir as a marker for neuronal activity,

and using experimental tooth movement to activate the trigeminal system, thalamic Fos-irwas located in intralaminar nuclei but not in the ventrobasal nuclei.489 The most probable

explanation for the absence of Fos-ir in the ventrobasal thalamus in the latter experiment

and ours is that neurons in this area are not able to exhibit Fos expression, which has beenreported before.43

All nuclei of the hypothalamus studied, exhibit augmented Fos expression after

capsaicin treatment, except for the LH. Direct projection pathways from trigeminal nuclei tothe hypothalamus (especially from the TNC and C1, C2 and within these nuclei in particular

from layer I, II and V) have been demonstrated,253 which could explain the reactivity to

nociceptive signaling of trigeminal nerves. The number of Fos positive cells in the 1000 nMcapsaicin treated group is generally higher compared to the 250 nM treated animals,

although significance is only reached in the SO. That neurons of the SO specifically react

dose-dependently to capsaicin fits with experiments that showed SO activation (measuredby discharge activity) in anaesthetized rats after noxious hind limb stimulation, just prior to

the respiratory and cardiovascular changes.140 Thus, it is likely that Fos expression in the SO

in our experiments is not the result of the physiological (respiratory, cardiovascular)responses to the pain, but it rather mediates these responses. Naturally, we cannot exclude

the possibility that the physiological responses to the pain indirectly modulate neuronal

activity in any of the nuclei studied. Actually, this is rather likely, as many neuro-endocrineand cardiovascular responses, initiated by nociception, feed back to the brain. Experiments,

like those performed by Hamamura and colleagues,140 may help in discerning direct from

indirect nociception-induced neuronal activity.DMH and VMH lesions induce the development of hyperalgesia after nociception,462

indicating that activation of these nuclei, or the neurocircuitry these nuclei are involved in,

inhibit the origination of hyperalgesia after nociception. Fos expression in the DMH and PVHcan be induced by restraint stress, but is further enhanced by a noxious visceral

stimulation.448 Electrical stimulation of the PVH induces analgesia393,493 and PVH lesions not

only significantly increases paw licking scores in the formalin test109 but also decreases theanalgesia after cold-water swim stress in the tail flick test.450 The latter suggests a role of

the PVH in stress-induced analgesia, but this may be dependend on the type of stressor, or

the type/location of pain, because the PVH is not involved in the stress-induced analgesiathat is induced after restraint in the formalin test.109

Forebrain Nuclei, cortical,A relative large proportion of the somatosensory cortex of the rat (66%) consists of

neurons that somatotopically represent the face480 and especially the whiskers. Of the


51

differential SI areas, in which we quantified Fos-ir, all layers of the upper lip region showed

significantly enhanced Fos expression in capsaicin treated rats compared to control rats.Capsaicin treatment enhanced Fos-ir in the SI jaw region was generally enhanced but not

significantly. The SI is mainly innervated by the ventrobasal thalamic complex (VPL and

VPM) and the Po.447 Especially the VPM and the Po receive projections from the marginallayer of the TNC389,390 and it is likely that nociceptive trigeminal information is relayed to the

SI through these nuclei. Projections from the VPM innervate layer 3, 4 and 6 of the SI

whereas the Po projects to layer 1 and 5a (reviewed in471). This may explain why the Fosexpression in all layers of the SI, upper lip region, is enhanced. As nociceptive neurons in

the SI are primarily located in layer V and VI,213,214 it is plausible that activation of layers V

and VI is caused by direct action of capsaicin on trigemino-nociceptive nerves. Fosexpression in the other layers most likely is caused indirectly, for example by the head

grooming and head scratching that is induced by the intracranial capsaicin.192,193 The latter

behaviour could also be involved in the significantly increased Fos expression in layer V andlayer VI of the SI, forelimb region after 250 and 1000 nM capsaicin respectively.

Quantitative differences of Fos-ir between 1000 and 250 nM capsaicin were

expected in parts of the cortex that are involved in autonomic and emotional control.However, the insular, cingulate and prelimbic cortex didn’t show any activation differences

between the 2 concentrations of capsaicin. Also, Fos-ir in the cingulate and prelimbic cortex

was not different between capsaicin and vehicle treated rats. Vehicle treated rats are alsosubmitted to (a mild form of) stress, caused by the novel environment rats are put into

during the intracisternal infusion. This may partly explain the Fos-ir in the cingulate and

prelimbic areas of control animals and the absence of differences between control andcapsaicin treated animals.

General discussionThis is the first report describing cerebral Fos expression patterns in conscious rats

after intracranial nociceptive trigeminovascular stimulation. In the first half of this century,

experimental intracranial nociceptive trigeminal stimulation in humans was performed tostudy trigeminovascular headaches320,354 but nowadays this is not acceptable due to ethical

reasons. The use of intracranial nociceptive trigeminal stimulation in conscious animals may

provide an alternative to mimic the pain of trigeminovascular headaches like migraine andcluster headache in humans. In humans, PET scan studies have revealed activation of

certain regions in the brain-stem and hypothalamus during migraine478 and cluster

headache133 respectively. Next to cortical (audiovisual, cingulate) regions, brainstem areasthat coincided with the LC and DR were active during the migraine attack, the latter

remaining active after abolition of the attack by sumatriptan, leading the authors to

conclude that these regions may be involved in generating the migraine attack.478 Theinferior hypothalamic gray is suggested to be involved in the pathophysiology of cluster

Chapter 2.2

52

headache as this region showed enhanced rCBF in cluster headache patients during thebout, but not outside the bout133 and this region is not activated in other types of headache,

like migraine478 or experimental head pain.279 Our results show that robust intracranial

nociception (1000 nM) is able to increase Fos expression in the LC, the DR, the DMD, theVMH, the PVH and the SO in conscious rats, implying that these areas may become

activated due to intracranial trigeminovascular nociception. These brain areas overlap with

the specific regions activated during cluster headache and migraine. Our data thus supportsthe possibility that hypothalamic and DR/LC activation during cluster headache and migraine

respectively is caused by the pain of the headache. Activation of the LC and the DR, 2 areas

well known for their role in anti-nociception,412 in combination with the action ofsumatriptan, may be necessary to completely block the pain of the migraine attack,

explaining the results of Weiller and colleagues.478 This also would be an alternative

explanation for the finding that sumatriptan is ineffective in aborting the headache of amigraine attack when given during the aura phase.20

Section 3

Immunesystem modulation oftrigeminovascular headache

Section 3

54

PREFACE

The first chapter in this section is a review about immunesystem function in migraine. The

comorbidity of migraine with atopic diseases like eczema and asthma, which are associatedwith dysfunction of the immunesystem, supports the hypothesis that migraineurs exhibit

altered immunesystem function. Migraineurs show increased susceptibility for infections,

benefit from eradication of H. Pylori infection and report that the headache is most intensewhen they suffer from infectious diseases. These examples provide indirect evidence for an

altered immunesystem in migraineurs. The review includes studies that have measured the

various components of the immunesystem in migraineurs. Thus, studies measuringimmunoglobulins, complement, histamine, cytokines and immune cells in migraineurs will be

considered.

The second chapter in this section describes the effects of inflammation on TNC I,II Fos

expression and behavioural responses in our conscious animal model of trigeminovascular

nociception. Enhanced sensitivity of trigeminal afferents caused by infection may explainwhy migraineurs report their headache of highest intensity when they suffer of infection.

Immunesystem in migraine

55

Chapter 3.1

Immune system function in migraine: a review1

SummaryStudies of the past 33 years that examined immunological parameters in

migraineurs are reviewed. Although there is no clear cut well defined immunological

disorder present in migraineurs, some immunological parameters are changed. These

include enhanced plasma histamine levels, increased spontaneous histamine release ofleukocytes, altered (possibly suppressed) lymphocyte function and interictally decreased

polymorphonuclear and monocyt phagocytotic capacity. These changes more likely point to

an underlying infectious disorder than to a recurrent atopic illness and agree with thefindings that migraineurs have an increased susceptibility for infections and that migraineurs

benefit from Helicobacter pylori eradication.

1 with: W.J. Meijler, J.Korf and G.J. Ter Horst. Submitted for publication in Journalof Neuroimmunology

Chapter 3.1

56

Introduction

Migraine is the most common neurological disorder in the human population,

affecting 6% of the male and 15-17% of the female population,418 but the pathogenesis ofmigraine is still largely unknown. Migraine is a complex syndrome as can be seen from the

various factors (foods, drinks, lack of foods, stress, stress relieve, menses related hormones,

environmental changes, smoking, exercise) that are reported to precipitate a migraineattack.7,34 The migraine attack is usually characterized by a severe, frequently occurring,

unilateral headache accompanied by nausea, vomiting and photo/phono-phobia. Prodromal

signs of migraine are changes in mood, alertness, appetite and fluid balance,32,143 which canbe present up to 24 hours before the headache phase. The 2 types of migraine most

migraineurs suffer from are classical and common migraine, the former differing from the

latter because it is characterized by an aura (visual, sensor and/or speech disturbances)occurring 15 to 30 minutes before the actual headache phase.

It is not known why selective precipitators cause migraine in some humans but not

in others. Physiological alterations rendering migraine patients vulnerable for theseprecipitators therefore are a topic in migraine pathophysiology research. Such a

physiological system should be different in migraine patients compared to non-migraineurs

and have the ability to increase the patients vulnerability for migraine triggers. This articleaims to show that parts of the immune system act different in migraineurs compared to

non-migraineurs and that these changes may render patients more vulnerable for migraine

precipitators.

Migraine: Hypersensitivity and InfectionsFour different types of hypersensitivity can be distinguished of which type I and III

may be of special relevance to migraine. In type I hypersensitivity, or allergy, the production

of IgE by plasma-cells plays a central role. IgE stimulates mast cells and leukocytes to

release mediators of inflammation like histamine. These mediators in turn cause vasodilationand plasma protein extravasation in small blood vessels, platelet aggregation and irritation

of sensory nerve terminals.445 Type III hypersensitivity is caused by large amounts of

immune complexes formed by, for example, circulating bacterial products. These immunecomplexes can stimulate the release of vasoactive amines from platelets and basophylic

granulocytes which results in platelet aggregation and recruitment of neutrophilic

granulocytes that cause local inflammation and damaging of the vascular wall. An exampleis the Arthus-reaction. Injection of antigen in the skin of a person with high antibody titers

causes a local inflammatory process that peaks between 4 to 10 hours after antigen

injection and disappears after 48 hours.445

The first argument for relating migraine to immune system dysfunction is the

observation of comorbidity of migraine and atopic disorders like eczema and asthma. Nelson


57

reviewed this issue in 1985, and concluded that a close association between migraine and

atopic diseases exists.317 Other studies confirm this association. One study, examining morethan 1000 children, found signifcant increased prevalence of migraine in children with atopic

diseases.308 Also, 6 % of children of mothers with migraine (but no asthma/allergies) had

asthma compared to 3.2 % of children of mothers without migraine and/or atopic diseasessuggesting a relation between the occurrence of asthma and migraine.59 Both disorders

appear in a paroxysmal and recurrent fashion and a hypersensitive response would well fit

with both the duration of the migraine attack and the fact that selective foods canprecipitate migraine. Based on these similarities it is understandable that migraine was

already linked to a hypersensitive immune system in 1913.231

More recently migraine has also been associated with infections. Migraineurs notonly report that infections precipitate their migraine attacks but also that headache is of

worst intensity during an infection.55 In migraine patients without aura, an elevated

frequency of (subclinical) infectious events (herpes labialis, pharyngitis, cystitis, vaginitis,mycosis) has been found.67 In 31 children with migraine, 29 had gastrointestinal

inflammation with oesophagitis, gastritis and/or duodenitis.276 Moreover, recently it was

shown that in a group of 81 migraineurs that are infected with Helicobacter pylori (H.pylori), 19 become free of migraine attacks (during a half year follow up period) after

successful H. pylori eradication. The remaining 62 migraine patients with successful

eradication show a marked significant reduction in intensity, duration and frequencycompared to 13 H. pylori infected migraine patients that received eradication treatment but

were not successfully eradicated from H. pylori.116 The report has to be confirmed by other

groups in the future, but it clearly shows the potential involvement of infectious diseases inmigraine pathophysiology.

The comorbidity of either atopic/allergic illnesses or infectious diseases

with migraine may be considered indirect evidence of the involvement of the immunesystem in migraine pathophysiology. This review will focus on studies that examined

different aspects of the immune system themselves in migraine patients to elucidate

whether it is indeed different in migraineurs and if different, whether these changes point toa hypersensitive and/or an infectious immune pathology.

MethodsArticles published from 1966 through 1998 were identified through a Silverplatter

Medline search. Keywords used were all discussed immune parameters (and abbreviation)

combined with the word migraine. Articles were selected if one or more of the immuneparameters were measured in a body fluid of migraineurs. The reference lists of selected

articles were then reviewed for additional relevant articles. An analysis of the reviewed

articles is depicted in table 1.

Chapter 3.1

58

Immunoglobulin EThe question whether migraine is a type I hypersensitivity reaction has extensively

been examined by the measurement of total IgE and food-specific IgE (IgE-s) in the serum

of migraine patients. It is important to distinguish whether there is a personal history ofatopy in migraine patients, as IgE elevations may be explained by comorbid atopic diseases.

Of ten studies examining IgE/IgE-s, 6 examined (indications of) atopy whereas one study

excluded patients with atopic illnesses from the experiments.316 In studies in which a rise ofIgE was found in migraineurs compared to normal levels, atopy is not examined163,299 or the

rise is correlated to atopy.29,97,284,345 The only study excluding atopic patients did not find a

rise of IgE in migraine patients,316 which was also found in 3 other reports.290,361,464 Thus inatopic patients, increased IgE levels may be associated with migraine whereas IgE is

probably not associated with migraine in non-atopic patients.

IgE levels are higher in symptomatic than in asymptomatic allergic patients.333

However, 9 out of the 10 studies examined, did not mention when the blood was collected.

It is likely that serum IgE levels were examined during the headache-free period because in

all the studies, IgE levels of migraineurs were compared to normal/control levels of non-migraineous persons. Accepting this, it seems that thus far, only one study examined IgE

levels in a few patients during the attack464 and found no changes of total IgE in

migraineurs compared to controls.Specific IgE types against migraine inducing foods (IgE-s) were found in some

migraine patients in 2 studies.299,345 Monro and co-workers found a high correlation between

the provoking food and serum IgE-s.299 Also 23 out of 26 patients responded withimprovement of the migraine after elimination of the IgE-s inducing foods from the diet.

However, in a challenge study of Pradalier and colleagues345 the IgE-s related food could

not induce migraine. Moreover, IgE-s could not be found in migraine patients in 2 otherstudies.290,316 Therefore, available data on serum levels of IgE-s are still controversial.

From these observations it can be concluded that a type I hypersensitive reaction may be

involved in migraine pathophysiology in atopic patients, but it is controversial whether or nota type I hypersensitivity is involved in the migraine pathophysiology of non-atopic patients.

HistamineIgE stimulates mast cells and leukocytes to release histamine. There is substantial

evidence that histamine is associated with migraine pathophysiology. Indirect evidence

derives from studies describing migraine and headache induction after histamine infusion inmigraine patients and control subjects, respectively.219,221,222 Histamine is a potent

vasoactive substance in many vascular beds.25,50,60,208,295,364,427,451 As the headache phase of

the migraine attack has been associated with vasodilation of cerebral arteries,166,374,446,485

histamine may be a candidate mediator of the vascular changes during a migraine attack.


59

Direct evidence of involvement of histamine in migraine pathophysiology has been

found in several studies. Studies from the early seventies did find changes in blood11 andurine398,399 histamine levels in migraineurs during and outside the attack compared to

controls. Also, the histamine metabolite 104MIA was elevated in urine of migraineurs

outside the attack.240

More recent studies could not confirm the changes in whole blood10,138,146 and

urine10,400,401 histamine levels in migraine patients. However, 2 studies described that

plasma histamine levels were higher in migraine patients both ictally and interictallycompared to non-migraineurs.138,146 Also, three other studies report higher spontaneous

histamine release (SHR) from leukocytes in migraineurs compared to non-migraineurs, both

interictally367,382 and ictally.383 In agreement with this, the latter study could not finddifferences in the SHR of leukocytes between the headache and headache-free phase in

migraineurs.383 Thus migraine patients have increased plasma levels of histamine that may

be related to the SHR of leukocytes, independent of the attack.It is not clear whether an other blood born factor than IgE is responsible for the

increased SHR of leukocytes in migraine patients. Initially, Selmaj and colleagues found

increased SHR by leukocytes of non-migraineous subjects after stimulation with migraineousserum382 but they could not replicate this in a follow-up study.383

A persistent infectious disease may be an alternative cause of chronically increased

plasma histamine levels. For example, Helicobacter pylori is well known for its stimulatingeffect on histamine release,195,269,347,348 especially of gastric mucosal cells, and has been

reported to produce histamine itself.461

Whereas anti-histaminergic treatment in migraine aimed at the H1 and H2 receptoris disappointing, the H3 receptor seems a promising anti-migraine target (reviewed by256).The H3 receptor agonist R(-)-α-methyl-histamine, as many other anti-migraine

drugs,45,48,262,365 has been found to effectively inhibit plasma protein extravasation (PPE)275

within the dura mater; a proposed pathophysiological phenomenon in migraine.

Other Immunoglobulins and ComplementCirculating immunoglobulins and complement are involved in types II and III

hypersensitivity and they are important in the lysis and opsonization of bacteria. In 1977,

Lord and co-workers found that in 9 migraine patients without aura, complement 4 (C4) andC5 was decreased in the early headache phase compared to the headache-free phase.242,243

Increased levels of IgA were found ictally and interictally in 20 classic migraineurs and IgA

and IgG levels were increased in 35 non-prodromal (common) migraineurs in both periods.A different study also found increased levels of IgA, IgG and also IgM during the headache-

free phase in migraineurs compared to controls392 but another study reported decreased IgA

levels interictally in migraineurs.177 The majority of the studies however failed to findchanges in IgA, IgG and IgM levels146,229,265,303,361,464 or complement factors28,303,464 in

Chapter 3.1

60

migraineurs. As measurements were performed both ictally and interictally28,146,303,464 theconclusion seems justified that there is no change in serum immunoglobulin or complement

levels in migraineurs both during and outside the attack phase. As a consequence there is

also no evidence for a major role of immunoglobulin/complement mediated type II or IIIhypersensitivity in migraine pathophysiology.

Also, these data do not support the increased presence of (possibly subclinical)

infections in migraineurs. However, before we can reject this, it may be necessary toexamine the expression of specific bacterial immunoglobulins in migraineurs and the

possible differential expression in healthy controls.

Immune cellsImmune cells are essential for both an infectious and a hypersensitive

immunological response. Basophilic and eosinophilic granulocytes (polymorphonuclear cells),monocytes, mast cells, natural killer cells and macrophages all are leukocytes that belong to

the innate or non-specific immune system. These cells react acutely to primary or repeated

contact with an antigen. Antigen presenting cells (macrophages, B-cells, dendritic cells)mediate communication of the non-specific immune system to the specific immune system

that consists of the B- and T-lymphocytes. The specific immune system reacts slowly on first

antigen contact but fast and effective at repeated contact with the antigen.445

The number of eosinophilic and basophilic granulocytes did not differ between

migraineurs and non-migraineurs both during the headache and headache free phase146 but

the phagocytotic capacity of these polymorphonuclear cells was found to be decreased inmigraineurs during the headache phase compared to non-migraineur.s66 The latter study

also reported a decrease of monocyte phagocytotic capacity ictally, together with a decrease

of monocyte counts ictally in migraineurs compared to controls.66 Moreover, a lack ofdifferences between the phagocytotic function of monocyte and polymporphonuclear cells

measured ictally and interictally was noted (not supported with data), implying a general

decrease of monocyte function in migraineurs. This was not confirmed in a more extensivestudy examining monocyte function in 110 migraine patients ictally. Gallai and co-workers

did find an increase instead of a decrease of the chemotactic response, phagocytoticcapacity, TNF-α / interleukin-1β production and respiratory burst of monocytes compared to

the attack-free phase.113 Interictally, only the chemotactic response was altered, and

decreased instead of increased, in migraine patients compared to controls.113 The study of

Gallai with approximately 5 times more patients and using IHS criteria points to increasedphagocytotic capacity of monocytes during the headache phase. These seemingly

contradictory results of Gallai113 and Covelli66 may be explained by the time of blood

sampling. Gallai and co-workers measured immune cell levels and function of all migrainepatients 2 h. after start of the attack. Covelli and colleagues on the other hand have

sampled all patients at a fixed moment of the day i.e. between 9 and 10 A.M. We conclude


61

from the observations that the increase in monocyte phagocytotic capacity during a

migraine attack as reported by Gallai is transient and present only at the beginning (after 2h.) of the migraine attack. Apart from this possibly brief increased monocyte phagocytotic

activity at the start of the attack, overall monocyte function (phagocytosis/chemotaxis) in

migraineurs is decreased compared to non-migraineurs. The generally decreasedphagocytotic capacity of monocytes and polymorphonuclear cells may be responsible for theincreased number of infections found in migraine patients.67 Lower monocyte levels of β-

endorphin interictally may be additional evidence for aberrant monocyte function inmigraineurs.22

The number of natural killer cells has been reported decreased in migraineurs (not

specified when the measurement was performed)121 and increased ictally in milk-inducedmigraine.265 However, most studies examining the quantity of natural killer cells could not

find changes in migraineurs.66,229,270 The quantity of various types of T-lymphocytes have

been reported increased during the headache-free phase compared to a milk-inducedmigraine attack in 6 patients.265 The total T-lymphocyte population was also increased by an

isosorbide dinitrate-induced attack,270 confirming these results. In spontaneous migraine

however, the total number of T-lymphocytes is not increased66,121,229 andcytotoxic/suppressor T-lymphocytes are rather decreased121,229 or unaltered66 than

increased.265 Other types and activation states of T-lymphocytes are not altered in

migraineurs.66,121,229,270

B-lymphocyte counts were increased interictally in migraineurs compared to

controls in one study229 but decreased in another.121 Three other studies did not find

changes in B-lymphocytes in spontaneous,66 milk-induced265 or isosorbide dinitrate-induced270 migraine attacks. Thus data concerning B-lymphocytes albeit not conclusive, do

not yield evidence for B-cell involvement in migraine pathophysiology.

Although the quantitive analysis of T- and B-lymphocytes numbers in migraineursdo not point to involvement of these cells in migraine, 3 separate studies examining certainqualities of lymphocytes in migraineurs suggest otherwise. Decreased β-adrenergic receptor

sensitivity,204 decreased β-endorphin levels230 and increased dopamine D5-receptor

expression19 were found in lymphocytes of migraine patients interictally compared to non-

migraineurs. These changes could be the result from pathophysiological changes occurring

elsewhere in the body, as suggested by all three studies. However the changes found maywell alter lymphocyte function. Dopamine has immunosuppressive actions that may beexerted through D5 receptors51 β-endorphin stimulates T-cell proliferation149,206 and

adrenaline both stimulates T-cell proliferation358 and alters natural killer cell activity oflymphocytes through β-adrenoceptors.147 The observed alterations in migraineurs thus point

to a reduced lymphocyte proliferative capability and enhanced sensitivity for dopamine to

exert its immunosupressive function.

Chapter 3.1

62

CytokinesCytokines not only mediate communication between the different immune cells but

also the communication between the immune system and other physiological systems of an

organism. Cytokines have been shown to induce headache,57,205,216,294,376,411 reviewed in405

but thus far few studies have examined cytokine levels in migraine patients. Compared tonon-migraineurs, migraine patients without aura showed higher serum levels of TNF-α70 and

of IL-1β69 (the latter only in 3 patients) interictally. Within migraineurs, plasma IL-1α, IL-1βand TNF-α were not elevated during the attack, but decreased levels of these cytokines

during the attack could not be excluded.458 The same report showed that body temperature

of migraine patients is significantly higher interictally compared to the attack phase.458 AsTNF-α can induce fever, the higher body temperature interictally also supports the

observation of increased plasma levels of TNF-α measured during the attack-free phase. In

food-induced common migraine, plasma IL-4 and IL-6 were decreased and plasma GM-CSFand IFN-γ were found increased ictally compared to the headache-free phase264 IL-6 is also

able to induce fever, which would coincide with the lower body temperature found in

migraine patients during the attack. Lower serum IL-4 levels during the attack were alsoobserved in isosorbide dinitrate-induced -and in spontaneous migraine.263 Finally, decreased

levels of IL-2 were found in migraineurs during the headache-free phase compared to non-

migraineurs.391

SummaryGenerally, immunoglobulins and complement do not seem to be altered in

migraineurs compared to non-migraineurs. Serum levels of IgE, important if migraine were

an allergic/atopic kind of disorder, has been extensively examined in migraine patients but if

increases are found it is probably related to comorbid atopic disorders. Thus, a causal rolefor IgE in migraine pathophysiology is uncertain. Plasma histamine levels and the

spontaneous histamine release of leukocytes are chronically increased in migraineurs.

Both cells of the specific and the non-specific immune system show altered function inmigraine patients. Polymorphonuclear cells show a decreased phagocytotic capacity and

monocytes have a generally decreased phagocytotic capacity which is enhanced only at the

start of the headache phase. There is evidence of decreased numbers of monocytes ictally.All studies examining lymphocyte qualities found evidence of aberrant (possibly suppressed)

lymphocyte function interictally in migraineurs compared to healthy controls. Increased TNF-α levels interictally and decreased IL-6 levels ictally agree with the observation that body

temperature of migraine patients is slightly decreased during the attack.

DiscussionSeveral comments on the studies that were reviewed have to be made. Most

studies only defined whether the measurements were done during or outside the headache


63

phase or did not specify this at all. However, all the studies that specified the time point of

measurement more precisely than during or outside the headache phase found changes inthe immunological parameter(s) studied.113,138,242,264,270,383 Mostly, one time point was

examined during the headache phase.113,138,264,270 Longitudinal studies however, conducted

during the headache phase,242,383 found transient effects, stressing the importance ofdefining an exact time point of measurement. This also raises the question whether the

absence of changes found in other studies is partly due to a ‘large’ variation in time points

of examination used for the individual measurements.Also, all reviewed articles examined systemic changes of certain immune

parameters. It has to be considered that local immunological dysfunction may be present in

migraineurs but this may be too small to induce any systemic changes. An example of sucha local immunological dysfunction that has been suggested to be associated with migraine is

neurogenic inflammation (NI) of the meningeal vasculature. NI is a process involving

vasodilatation and plasma protein extravasation (PPE) that is caused by afferent release ofneuropeptides like Calcitonin Gene Related Peptide (CGRP) and Substance P (SP).311 It has

been shown that after trigeminal afferent stimulation in rats, mast cell degranulation occurs

in the dura mater87 and although it is not likely that mast cell degranulation is involved inthe PPE,260 it may contribute to the inflammatory process in other ways. The anti-migraine

drugs Sumatriptan,45 classic ergot alkaloids365 and Naratriptan64 inhibit dural PPE elicited by

trigeminal afferent stimulation in animal models. Furthermore, the efficacy of non-steroidal-anti-inflammatory-drugs in inhibiting dural PPE48 and migraine327,342 argue for the relevance

of NI in migraine as do the reported elevated CGRP levels in jugular vein plasma samples of

migraine patients during a migraine attack.128

However, elevation of SP could not be found in the same patients,128 which may

provide arguments against NI. Moreover, inflammation of the meninges has never been

detected in migraineurs. Most anti-migraine drugs not only ameliorate PPE but also inducevasoconstriction. Bosentan, which blocks PPE without having vasoconstrictive effects, was

ineffective in alleviating migraine attacks, when given during the headache phase,277

implying that NI is not involved in migraine. However, it cannot be excluded that NIprecedes the headache phase of migraine. Therefore, treatment with drugs like Bosentan

before the actual headache phase would be necessary to definitely prove the irrelevance of

NI in migraine.NI thus remains a possible mechanism for the pathophysiology of migraine. Migraine

develops from the autonomic disturbances in the pro-dromal phase into the severe

headache phase approximately 24 h. later. A developing sterile inflammatory process in thecerebrovasculature could well fit within this process. The finding that stimulation of the

(parasympathetic) sphenopalatine ganglion-induced trigeminal afferent dependent PPE in

the dura mater of rats15 indicates that autonomic misbalance, and overactiveparasympathetic function in particular, might indeed induce neurogenic inflammation.

Chapter 3.1

64

A different local immunological involvement in migraine pathophysiology isprovided by the report that spreading depression (which is a proposed pathophysiologicalmechanism for the aura phase of migraine223) induces TNF-α expression in meningeal

vasculature.58 As TNF-α might induce the release of nitric oxide486 which is known to induce

migraineous headache in migraineurs168,220 it may be involved in the headache phasefollowing the aura. Moreover, Worrall et al have shown that TNF-α may induce PPE,486

which possibly also occurs during/before a migraine attack.Immune system-induced hyperalgesia is a possible physiological mechanism

rendering patients more vulnerable for migraine. It has been shown in several animal

models that immune-activation can induce hypersensitivity of different nociceptive nerveslocated extracranially,326 in tracheal perfusates,161 in the skin153 and in the hind paw of

rats.74,104 As intracranial trigeminal nerves are most likely involved in the pathophysiology of

migraine and headache in general, we demonstrated in a model of intracranial trigeminalstimulation in conscious rats192 that intracranial trigeminal nerves also show sensitization

after immunological stimulation with lipopolysaccharides.193 Immunesystem-induced

hyperalgesia could explain why headache in migraineurs is of highest intensity after aninfection55 but also why some people react with migraineous headache to certain

precipitators while others do not.


65

ConclusionThere is no clear-cut well defined immunological disorder present in migraineurs.

Results are conflicting and therefore a well-planned study in this field might be usefull.

Most studies showed that IgE and other immunoglobulins are not altered in

migraineurs (and if altered, probably it is related to comorbid atopic disorders), and thatplasma histamine levels are chronically elevated. A recurrent atopic/hypersensitive disorder

is therefore unlikely.

Migraineurs have an increased susceptibility for various infections and benefit fromeradication of Helicobacter pylori. The decreased polymorphonuclear and monocytephagocytotic capacity interictally and the increased plasma levels of TNF-α and histamine

found in migraineurs fits with this increased susceptibility for infections. These changes arenot attack related but rather chronically present and are therefore most likely not involved in

initiation of the attack. The inflammatory response, however, could induce hyperalgesia of

intracranial nociceptive afferents, rendering migraineurs vulnerable for certain precipitators.

Chapter 3.1

66

Table 3.1.1. Analysis of articles that measured immune parameters in migraineurs.

Complement / immunoglobulins Substance Com-

partment nr. Type of migraine Disease

Criteria Subjects studied Disease state Change in Migraineurs

or during attack Reference

Classical (21), IgE↑ in 0/21 IgE Serum 67

Common (46)

Spec. NIH, Atopy: in 18/67.

Migraine vs. normal (not specified)

Not specified

IgE↑ in 4/46

284

Early headache phase

C4↓ , C5↓ , -

9 Headache vs. headache-free, paired

Late headache phase

-

C3 bdp in 3 of 9, related to subsequent attack

Early headache phase

C4↓ , C1s↓ , C1(1)↓, -

Bf, C1q, C1s, C1(1), C3, C3 bdp, C4, C5,

18

Non-prodromal migraine

Headache vs. headache-free, not paired Late headache

phase -

74 All types Headache vs. headache free

N.S. -

20 Prodromal Migraine IgA↑

35 Non-Prodromal - Common

IgA↑, IgG↑

IgA, IgG, IgM

Serum

19 Non-Prodromal – focal neurologic symptoms during headache

Spec. A, Atopy: Immediate hypersensitive reaction patients included

Migraine vs. control Either during headache or headache free periods. IgA↑, IgG↑ ,IgM↑

242,243

IgE, IgE-s Serum 33 N.S., Dietary (23) and non-dietary (10)

N.S., atopy: N.S.

Migraine vs. normal N.S. IgE-s↑ in dietary migraine, -

299

C3, C4, IC, ANA, ADS-DA

ANA in 8/30 patients observed, -

IgA, IgG, IgM

Serum 30 Classical (10) or Common (20)

Spec. NIH, atopy: excluded.

Headache vs. headache free

Headache free sample: no headache at least 24 hrs. before or after sample. Headache sample at various times

-

303

40 Classical (10), Common (27), Cluster (3)


During headache free period

- C1q, C3, C3 bdp C4, C7, CH50, Bf

Blood, not further specified 10 Classical or common

N.S., atopy: N.S.


Before, during and after an attack

-

28

12 Headache -

18

Migraine vs. control

Headache free (for 5 days)

-

IgA, IgG, IgM

Serum

18

N.S. N.S., Atopy: 4 with non active hay fever, No drugs

Headache vs. headache free. Paired (12) and unpaired

Headache free for 5 days

-

146

IgE, IgE-s Serum 64 Children with severe migraine (44% prodromal, 56% common)

Spec. D, Atopy: 55% with atopic diseases.

Migraine vs. normal (>150 IU/ml)

N.S. IgE↑ in 18/64, - 97

C3, C4, CH50

C3↑ , -

IgA, IgG, IgM

Blood, not further specified

54 Classical (15) or common (39)

Spec. NIH, Atopy: N.S.

Migraine vs. control During headache free period

IgA↓, -

177


67

Complement / immunoglobulins continued… Substance Com-




IgE, IgE-s, IgG4

Serum 119 Classical (60) or Common (59) / Dietary (74), Non-dietary (45)

Spec. Vahlquist, Atopy: 20% IgE against atopic allergens.

Migraine vs. control Dietary vs. Non Dietary migraine

N.S. - 290

IgE, IgE-s, Serum 50 Common Spec. NIH, Atopy: included


N.S. IgE↑ in 7/50 ! 4/7 atopy, IgE-s↑ in 6/50

345

IgE, IgE-s Blood, not further specified

12 Children with Abdominal migraine

Spec. C, Atopy: included


N.S. IgE↑ in 4/12 ! 4/4 atopy, -

29

IgA, IgD, IgE, IgG, IgM

Serum 6 Common (6), drug free for 2 weeks


Migraine vs. control N.S. - 361

Bf, C1q, C3, C4, CH50

-

IgA, IgE, IgG, IgM

Serum 44 Classical (12) or Common (32)

N.S., Atopy: N.S.

Migraine vs. control During headache (16) and/or during headache free period (44). -

464

IgE Serum 49 Not specified N.S., Atopy: N.S.

Migraine vs. normal (250ku/l)

N.S. IgE↑ in 38.8% 163

CIC 21 Food related migraine, no atopy symptoms

Migraine vs. control Headache free phase

CIC↑

4 h. after challenge

- IgG4, a-IgG ab.

Serum

6 Food related migraine, no atopy symptoms, Migraine induced by milk challenge

Spec. IHS, Atopy: excluded

Headache vs. headache free (sample at t=0 h. direct before food challenge)

72 h. after challenge -

265

IgE, IgE-s Serum 105 Common migraine Spec. NIH, Atopy: excluded

Migraine vs. Normal (N.S.)

N.S. - 316

IgA, IgG, IgM

Serum 40 MWA, drug free for 14 days

Spec. IHS, Atopy: excluded

Migraine vs. control Headache free IgA↑, IgG↑ , IgM↑ 392

IgA, IgG, IgM,

Serum 12 MWA (non food-induced), drug free for 20 days

Spec. IHS, Atopy: N.S.

Migraine vs. control Headache free for at least 2 days

- 229

Chapter 3.1

68

Histamine Substance Com-




Histamine, 1-4MIA

Urine

32 N.S. N.S. Migraine vs. normal Headache free 1M4IAA↑ 240

Migraine vs. Control (from other studies)

Headache free phase, 24 h. samples

Histamine↑ Histamine Urine 11 Classical and common migraine

N.S., Atopy: N.S.


24 h. samples Histamine↓

399

Pre headache vs. headache, 24 h. samples

Histamine↑ Histamine Blood 10 N.S. N.S., Atopy: N.S.


Post headache vs. headache 24 h. samples

-

11

Histamine Urine 5 Classical (4) Common (1)



4 periods of a attack day (0-4-8-12-24 h) vs. same periods of attack free day (at least 2 days later)

Histamine ↑ during attack free period in 2 patients

398

Urine

-

Histamine

Blood

10 N.S. N.S., Atopy: N.S., at least 3 days drug free


Headache free samples before and after the headache

-

10

14C-histamine

-

14C-histamine metabolites

Urine 2 N.S. Spec. NIH, Atopy: N.S., drugfree during study

Migraine vs. Control (from other studies)

During headache phase

-

400

Histamine 19 Day of attack vs. attack-free day.

-

Histamine

Urine

9

Approx. 2/3 Classical

N.S., Atopy: N.S., no drugs during study


4 periods of a attack day vs. 4 periods of attack free day (paired)

-

401

SHR Leuco-cytes

9 Common (3), Classical (1), Cluster (1), Basilar Artery (3) and Cyclic vomitting + frontal headache (1)

Spec. B, Atopy: N.S., no food allergy., 7 on medication

Migraine vs. control Headache free (at least for a week) phase

SHR↑ 367

12 Headache - Blood 18 Headache free (for

5 days) -

12 Headache Histamine↑ 18



Histamine↑

Histamine

Plasma

18

N.S. N.S., Atopy: 4 with non-active hay-fever., No drugs


Headache free for 5 days. Paired (12) and unpaired

-

146

SHR Leuco-cytes

17 Common Spec. NIH, Atopy: excluded., drug free for 1 week.

Migraine vs. control Headache free (at least 3 days) phase

SHR↑ 382


69

Histamine continued … Substance Com-




Prodromal period (3)

SHR↓

First 3 h headache (8)

SHR↑

3-12 h headache (8) -


>12 h headache (8) -

SHR Leuco-cytes

27 Common (22), Classical (5)

Spec. NIH, Atopy: N.S.,

Headache vs. Headache free

First 3 h headache (8)

-

383

3 N.S., migraine provoked by food challenge

Histamine↑ Histamine Plasma

2 Placebo challenge

Spec. E, Atopy: N.S.


After challenge

-

257

Blood -

Plasma

18 Headache free

Histamine↑

Blood -

Histamine

Plasma

9

Common Spec. Blau, Atopy: N.S., drug free for at least 2 days


First 2 h. headache

Histamine↑

138

Chapter 3.1

70

Immune system cells Type of cells Property Nr. Type of migraine Criteria Subjects studied Disease state Change in Migraineurs


12 Headache -

18



-

Bas, Eos Counts

18

N.S. N.S., Drugfree

Headache vs. headache free (paired (12) and unpaired)

Headache free for 5 days

-

146

tT, hT, cT, T+, B, NK, Lympho-cytes, Monocytes

Counts 36 Classical (11) Common (25)

NIH, IHS, Saper

Migraine vs. control and chronic tension type headache

N.S. B↓, NK↓ , cT↓ , - 121

Lympho-cytes.

β-adrener-gic sensiti-vity

12 MA (1) MWA (11)

IHS Migraine vs. control Headache free β-adrenergic sensitivity ↓

204

4 h. after challenge

tT↑ , cT↑ , eT+↑ , mT+↑, K/NK↑ , -

K/NK, tT1, hT, cT, eT+, mT+, B/T+

Counts 6 Food related migraine, no atopy symptoms, Migraine induced by milk challenge

IHS Headache vs. headache free (sample at t=0 h. direct before food challenge)

72 h. after challenge eT+↑ , -

265

Migraine vs. control Headache (samples between 9 and 10 A.M.)

Phagocytosis ↓ PMN, Monocytes

Phagocy-tosis (of Candida albicans)

23


samples between 9 and 10 A.M.

-

tT, hT, cT, Monocytes, NK, B,

Counts 18

Common, no atopy NIH., drugfree

Migraine vs. control Headache (samples between 9 and 10 A.M.)

Monocytes↓ , -

66

MA (27) β-endorphin levels↓ Lympho-cytes

β-endor-phin levels

87

MWA (60)

IHS, drugfree for 2 weeks

Migraine vs. control Headache free (for at least 24 h.)

β-endorphin levels↓

230


71

Immune system cells continued… Type of cells Property Nr. Type of migraine Criteria Subjects studied Disease state Change in Migraineurs


MA (40) Chemotactic response ↓

MWA (70)

Migraine vs. control Headache free (for at least 7 days)

Chemotactic response ↓

MA (40) Chemotactic response ↑

Chemo-tactic response

MWA (70)


Headache sample 2 h. after start attack. Headache free (for at least 7 days)

Chemotactic response ↑

MA (40) -

MWA (70)

Migraine vs. control Headache free (for at least 7 days) -

MA (40) Phagocytosis ↑

Phago-cytosis (of Candida albicans)

MWA (70) Headache vs. headache free


Phagocytosis ↑

MA (40) -

MWA (70)


MA (40) TNF-α, IL-1β production ↑

TNF-α, IL-1β produc-tion

MWA (70)



TNF-α, IL-1β production ↑

MA (40) -

MWA (70)


MA (40) Respiratory burst↑

Monocytes

Respira-tory burst

110

MWA (70)

Spec. IHS, drug-free (for 20 days)



Respiratory burst↑

113

tT1, tT2, hT, cT, T+, B, Monocytes, NK

Counts 12 MWA (non food-induced), drug free for 20 days

Spec. IHS Migraine vs. control Headache free (for at least 2 days)

B↑, cT↓ , - 229

tT1, B, NK, hT, cT.

Counts 22 MWA, migraine induced by isosorbide dinitrate

Spec. IHS Headache vs. headache free

Headache sample 90 minutes after isosorbide dinitrate challenge

tT1 ↑ , in controls (5) increase is absent, -

270

Lympho-cytes

D5-receptor expres-sion

11 MWA(8) MA(3), no prophylactic drugs

Spec. IHS Migraine vs. control Headache free (for at least 72 hours)

D5-receptor expression↑

19

Monocytes β-endor-phin concen-tration

13 MWA Spec. IHS Migraine vs. control Headache free (for at least 48 hours)

β-endorphin concentration↓

22

Chapter 3.1

72

Cytokines Substance Com-




IL-4, IL-6, IFN-gamma, GM-CSF

Plasma 14 MWA., Migraine induced by food challenge

Spec. IHS Headache vs. headache free

Headache free sample direct before food challenge. Headache sample 4 hrs after food challenge.

IL-4↓ IL-6↓ IFN-gamma↑ GM-CSF↑

264

TNF-α Serum 20 MWA Spec. IHS, drug free for 3 at least 3 weeks

Migraine vs. Chronic tension type headache or control

Headache free phase

TNF-α↑ 70

Serum IL-1β↑ IL-1β

Mononu-clear cells after LPS stimulation

3 MWA Spec. IHS Migraine vs. control Headache free phase IL-1β↑

69

Plasma 20 - IL-1α, IL-1β TNF-α Blood,

after LPS stimulation

20 MA (6) MWA (14)

Spec. IHS, drugfree


Attack and attack free sample at the same time of day (15) or attack free 5-7 h. later (5)

-

458

IL-2 Serum 13 MWA Spec. IHS Migraine vs. control Headache free phase

IL-2↓ 391

20

MWA induced by isosorbide dinitrate

Migraine vs. control (also challenged with isosorbide dinitrate

IL-4

Serum

10 MWA

Spec. IHS


Headache phase IL-4↓

263


73

Used Abbreviations:

Immunoglobulins and complement

ADS-DA Anti double strained DNA antibodiesANA Anti nuclear antibodiesBf Factor BC1q q fragment of Complement 1C1s s fragment of Complement 1C1(1) Complement 1 inhibitorC3 Complement 3C3bdp Complement 3 breakdown productsC4 Complement 4C5 Complement 5C7 Complement 7CH50 Total complement levelCIC Circulating Immune ComplexesIgA Immunoglobulin AIgD Immunoglobulin DIgE Immunoglobulin EIgE-s Immunoglobulin Especific against a type of food allergenIgG Immunoglobulin GIgG4 Immunoglobulin G4IgM Immunoglobulin M

Cytokines

IFN-gamma Interferon gammaIL-1 Interleukin 1IL-2 Interleukin 2IL-4 Interleukin 4IL-6 Interleukin 6GM-CSF Granulocyte / Macrophagecolony stimulating factor

Histamine

1-4MIA 1-methyl-4-imidazole acetic acid,main metabolite of histamineSHR Spontaneous histamine release

Immune system cells

B B-lymphocytestT1 total T-lymphocyte populationtT2 total T-lymphocyte populationplus a subset of NK cellshT helper/inducer T-lymphocytescT cytotoxic-suppressor T-lymphocytesT+ T-activated cellseT+ early T-activated cellsmT+ mature T-activated cellsB/T+ B cells and T-activated cellsK/NK Killer and Natural Killer cellsBas Basophilic cellsEos Eosinophilic cellsPMN Polymorphonuclear cellsD5-receptor Dopamine D5-receptor

Articles use different denotations for the different typesof lymphocytes that are measured. For legibility of thetable, some denotations (like CD3+ cells) have beentranslated into others (like total T-lymphocytepopulation).

General

- No further differences foundLPS LipopolysaccharidesMA Migraine with AuraMWA Migraine without AuraN.S. Not specified

Subjects Studied

Control A specified defined control group in the same study,unless otherwise noted

Normal Values from other studies or unpublished data thatdescribe a ‘normal’ range of the measuredimmunological parameter

Disease stateThe various articles use different terms to describe the diseasestate of migraine patients. For legibility of the tables, descriptionslike attack or symptom free where translated into headache phaseor headache-free phase respectively, although the authorsacknowledge that these are actually different terms.

Criteria:Spec. A Criteria migraine: severe or throbbing

headache, periodic, unilateral, nausea orvomiting or focal neurologic symptoms. Alsobilateral headache if photophobia and nauseawere present.

Spec. B Classic migraine: with aura and unilateral;Common migraine: no aura, throbbing,photophobia, nausea; Basilar artery migraine:ataxia, vertigo, nystagmus, vomiting.

Spec. C Criteria migraine: recurrent abdominal pain forminimum of 3 months, nausea/vomiting,family history of classical migraine, pallor.

Spec. D Criteria migraine: Headaches at least 1 a weekfor past 6 months, with 2 of the followingsymptoms: pallor, nausea, abdominal pain,photophobia, visual disturbances, giddiness,weakness and/or paresthesia down on oneside of the body.

Spec. E Criteria migraine: Unilateral start – radiatingbilateral, throbbing, temporal or occipitaldistribution, nausea, ipsilateral visual blurring,improvement by isometheptene orergotamine.

Spec. Blau Criteria according to Blau33

Spec. IHS Criteria according to the Ad hoc committee onthe classification of headache of the IHS145

Spec. NIH Criteria according to the Ad hoc committee onthe classification of headache of the NIH2

Spec. Saper Criteria according to Saper370

Spec. Vahlquist Criteria according to Vahlquist457

Chapter 3.1

74

LPS induced hyperalgesia

75

Chapter 3.2

Lipopolysaccharide-induced hyperalgesia of intracranial capsaicin sensitiveafferents in conscious rats1

SummaryMigraineous and non-migraineous headache is reported of highest intensity after aninfection. This study investigated whether activation of the immune system can induce

hyperalgesia in intracranial capsaicin sensitive afferents. The effects of intraperitoneal

injected lipopolysaccharides (LPS) on behaviour and Fos expression in the trigeminal nucleuscaudalis layer I, II (TNC I,II) elicited by intracisternally applied capsaicin was studied. Low

concentrations of LPS potentiated capsaicin-induced immobilization behaviour without

affecting Fos expression in the TNC I,II. High amounts of LPS however increased thenumber of capsaicin-induced Fos positive cells in the TNC I,II. These effects of LPS on

capsaicin sensitive afferents are probably mediated by cytokines that act at peripheral vagal

nerves, central brain regions or via direct actions of cytokines on capsaicin sensitive afferentnerve terminals. The hyperalgesic action of LPS on intracranial trigeminal and possibly other

capsaicin sensitive afferents of the head may explain why different types of infections are

accompanied by headache and why migraineous and non-migraineous headache is ofhighest intensity after an infection.

1 with: M.B. Spoelstra, W.J. Meijler and G.J. Ter Horst. Published in Pain, 78 (1998) 181-

190.

Chapter 3.2

76

Introduction

Stress, fatigue and menstrual periods are well known precipitators of migraine.

Less well known is that infections also can precipitate migraine and that infections giveheadache of highest pain intensity in both migraineurs and non-migraineurs when compared

to pain induced by other precipitators.55 There is also evidence that the immune system of

migraine patients is different compared to non-migraineurs.66,68-70 It is not clear howinfections trigger migraine or how they can enhance pain intensity. A possible explanation

for increased pain intensity is that intracranial trigeminal sensory nerves become

hyperalgesic after an immunological challenge. This induction of hyperalgesia bycomponents of the immune system has been shown for several peripheral sensory nerves.Cytokines like tumor necrosis factor alpha (TNF-α), interleukin 6 and interleukin 1-β (IL-1β)

are able to induce hyperalgesia in a nociception model that uses mechanical stimulation ofthe hind-paw of the rat104.74,104 Injection of these cytokines reduced the reaction time for

rats to respond to mechanical pressure applied to the hind paw. Moreover capsaicin-induced

vasodilatation in the rat skin, which is thought to be mediated by nociceptive afferents,could be enhanced by IL-1β.153 Furthermore, lipopolysaccharides (LPS, LPS are endotoxins

of the cell wall of gram-negative bacteria) could facilitate the release of calcitonin gene

related peptide (CGRP) from capsaicin sensitive sensory nerves located in the trachea ofrats.161 Cytokines like IL-1ß and TNF-α are critically involved in this facilitation.

None of the above mentioned studies examined effects of inflammation on

intracranial trigeminal sensory nerves. Trigeminal sensory nerves are the primarynociceptive afferents of the head and are generally considered to be involved in

headache/migraine pathophysiology.47,125,309 IL-1ß has been shown to increase nociceptive

processing in the trigeminal nucleus caudalis326 but these experiments used extracranialtrigeminal nerve stimulation in anaesthetized rats. The above mentioned observations

prompted us to examine whether intracranial sensory nerves are also subject to

sensitization after an immunological challenge. This might explain enhanced headacheintensity after infection seen both in migraineurs and non-migraineurs.55 Intracisternal (i.c.)

infusion of the irritant capsaicin in conscious rats was used to activate trigeminal nociceptive

fibers.192 A low and high dose of intraperitoneally (i.p.) injected LPS was used to stimulatethe immune system of the rat. LPS delivered systemically mimics many aspects of bacterial

infection including immunological alterations307 fever and pain.476 Capsaicin-induced Fos

protein expression in the outer layers of the trigeminal nucleus caudalis (TNC I,II) wasquantified to assess activity of the nociceptive part of the sensory trigeminal system.

Behaviour shown during and directly after infusion of capsaicin was recorded on videotape

and analyzed after the experiment. The Fos expression in the nucleus of the solitary tract(NTS) and area postrema (AP) was also quantified.


77

MethodsAnimals

Male Wistar rats weighting 308 ± 4.1 gr. were used. All rats were housed group

wise (3 rats/cage) on a light/dark regime (L/D: 08:00 h / 20:00 h) and surgery was

performed 5 days after arrival.Experiments were approved by and under close supervision of the committee on

Animal Bio-Ethics of the University of Groningen (FDC 1191) and performed according to the

ethical guidelines for investigations of experimental pain in conscious animals.503 Based onprior experiments192 in which the 100 nM capsaicin concentration did not significantly

augment the Fos expression in the TNC I,II and 1000 nM gave a maximal activation, we

chose to use a 250 nM capsaicin concentration. As rats were sacrificed 2 h. after capsaicintreatment, it was ensured that the pain caused by capsaicin is short-lived. None of the

animals suffered to such extend (behavioural observations) that they had to be terminated

before the end of the experiments.

Surgical proceduresCannula's, surgical materials and rat skin were disinfected with 0.5% chlorhexidine.

All rats were anaesthetized with 0.4 ml/kg i.m. hypnorm (fentanyl 0.3mg/ml and fluanisone

10mg/ml; Janssen, Beerse, Belgium) and pentobarbital (24 mg/kg i.p.). A midline incision in

the skin at the top of the head was made and membranes from the parietal, interparietaland rostrodorsal part of the occipital skull were removed.

The Cisterna Magna (CM) cannula was prepared from a stainless steel needle

(0.6x25 mm, 23G x 1"; Braun, Melsungen, Germany) which was shortened to 6.5 mm. Ratswere placed in a stereotaxic apparatus with incisor bar at –7 mm from the horizontal plane.

Two holes were drilled into the caudal corners of the interparietal skull and 2 screws (d. 1.0

mm, l.3 mm) were driven 1.5 mm into the skull. A hole (d. 1.2 mm) was drilled at themidline of the external occipital crest for placement of the CM cannula. The CM cannula was

carefully placed through the hole with a horizontal rostro-caudal approach and pushed

beneath the dorsal part of the occipital bone until the dorso-caudal part of the occipital bonewas reached. Then the cannula was slowly turned from the horizontal, rostral-caudal plane

into the dorsal-ventral plane. Guiding the CM cannula along the occipital bone caudal from

the cerebellum it was gently positioned into the Cisterna Magna. Correct placement of thecannula was confirmed by withdrawal of CSF after which the cannula was fixed to the skull

with dental cement (Kemdent, Purton Swindon, UK) and closed with a piece of silicon tube.

The wound was sutured and rats were allowed to recover for 3 days.

Experimental procedures A time line drawing for the experiments is shown in figure 3.2.1.

Chapter 3.2

78

InjectionFive hours prior to the infusion of capsaicin or vehicle rats were injected

intraperitoneally with either vehicle, 0.75 mg/kg LPS (LPS(L)) or 37.5 mg/kg LPS (LPS(H)).Concentrations of LPS and the period of 5 hours were based on the study of Hua and

colleagues who showed that 0.75 mg/kg LPS enhanced capsaicin-induced CGRP release

from sensory nerves of the trachea 5 h. after LPS injection 161. The higher concentration of37.5 mg/kg LPS was used to study possible dose dependent effects of LPS.

InfusionRats were placed into the experimental cage (30, 30, 30 cm) and capsaicin (250

nM) or vehicle was infused into the CM with a microinjection pump (CMA100, CarnegieMedicin, Stockholm, Sweden). Rats received 100 µl capsaicin in 2 minutes. During the

infusion and 10 minutes thereafter rats were filmed on videotape to allow analysis of

behaviour afterwards.

Perfusion and immunocytochemistryRats were perfused 2 h. following infusion of capsaicin or vehicle. Prior to the

transcardial perfusion rats were deeply anaesthetized with sodium pentobarbital andperfused with 0.9% saline for 1 min, followed by 4% paraformaldehyde (PF) in 0.1 M

phosphate-buffered saline (pH 7.4) for 20 min. After removal of the occipital bone,

placement of the cannula in the CM was confirmed and extent of the infusion into theepidural space was determined by inspection of the Evans Blue (dissolved (0.2%) in the

capsaicin and vehicle solutions) staining. After the removal, the brains were post-fixed in

4% PF during 24 h. Brain stem and upper spinal cord were cryoprotected by overnight

Day 0: Day 3:

t=0h.Injection:Saline,LPS(L),LPS(H)

t=5h. Infusion:Vehicle,

Capsaicin

t=7h.Perfusion

Surgery

Figure 3.2.1: Time line drawing of the experiments. A cisterna magna cannulawas implanted 3 days prior to the day of experimenting. At day 3 rats were i.p.injected with saline, 0.75 mg/kg LPS (LPS(L)) or 37.5 mg/kg LPS (LPS(H)). Aftert=5 h. rats were infused with vehicle or 250 nM capsaicin for 2 minutes.Behaviour was recorded during infusion and the 10 minutes afterwards. Two h.after infusion rats were perfused


79

storage in 30% sucrose in 0.1 M phosphate buffer (pH 7.4). Forty µm thick coronal serial

sections were prepared on a cryostat microtome at -15°C, and collected in 0.2 Mpotassiumphosphate-buffered saline (KPBS, pH 7.4) with sodiumazide (0.1%).

Free floating sections were immunocytochemically stained for Fos protein accordingto the following protocol. Sections were rinsed 3x10 min. in KPBS, pre-treated with 0.3 %

H2O2 in KPBS for 10 min, rinsed 3x10 min. in KPBS and pre-incubated in 2% bovine serum

albumin (BSA; Merck, Darmstadt, Germany), 2% normal serum (NS, normal rabbit serumSigma Chemie, Bornem, Belgium) in KPBS for 4 h. at room temperature. Subsequently,

sections were incubated in 2% BSA, 2% NS and primary antibody sheep-anti-Fos (1:2000;

Cambridge Research Chemicals, Northwich, UK) in KPBS with 0.5% triton X-100 (KPBS-T;Bayer, Deventer, Netherlands) overnight at room temperature. Sections were rinsed 3x10

min. in KPBS and incubated in 2% BSA, 2% NS and secondary antibody (1:200 biotinylatedrabbit-α-sheep IgG (Pierce, Rockford)) in KPBS-T at room temperature for 2 hours. After

3x10 min. washes in KPBS, sections were incubated in avidine-biotine-peroxidase complex

(Vector Labs, Burlingame) in KPBS-T with 2% BSA for 2 h. at room temperature. Hereafter,

sections were washed in 3x10 min. KPBS and 2x10 min. in 0.1M sodiumacetate buffer(NaAc, pH 6.0). For the final staining procedure 3.3'-diaminobenzidine tetrahydrochloride

(0.05%) and ammoniumchloride (0.04%) were dissolved in 1/2 v distilled water and 1/2 v

NAS solution (5% NikkelAmmoniumSulfate dissolved in 4/5 v 0.2M NaAc and 1/5 v distilledwater). To start the diaminobenzidine reaction 0.3% H2O2 was added. The reaction was

stopped after 20 minutes. Sections were washed 2x10 min. in 0.1M NaAc and 3x10 min. in

KPBS, mounted on gelatin coated slides, air dried, dehydrated in graded ethanol's and xyloland cover-slipped with DEPEX. All staining procedures were with gentle agitation.

QuantificationTNC layer I, II.

Fos immunoreactive cells were counted at -1, -2, -3, -4, -5 and -6 mm caudal from

obex by an observer blinded from experimental procedures. Sections from -0.5 to -1.5 mmwere averaged to obtain the count for the -1 mm level and so on. To obtain accuratesampling of sections for each level, the trigeminal nucleus of one rat was dissected (40 µm,

freezing microtome) from obex to -7 mm from obex and all sections were immediatelymounted on gelatin coated slides. A Nissl staining was performed to show cytoarchitecture

of the sections and the Fos stained sections were compared to these sections to determine

exact distance of the Fos expression from obex. Because there were no significantdifferences in the number of Fos positive cells between the right or left side of the TNC I,II,

the total number of cells per section was counted. The mean of the total TNC I,II was

calculated by averaging the Fos expression at the 6 levels.

NTS and AP

Chapter 3.2

80

Because of the functional differences between both the medial/lateral and therostral/caudal parts of the nucleus of the solitary tract (NTS), Fos positive cells were

counted in the lateral and medial divisions of the NTS at the level of the obex (bregma –

13.68 mm) and also more rostrally at bregma –11.6 mm.335 The Area Postrema (AP) wascounted at the level of the obex (bregma –13.68 mm335). Three sections of each part of the

NTS and AP were counted and averaged for each animal.

Weight-loss and temperatureTo assess whether the different concentrations of LPS had an effect on body-

weight or temperature rats were weighed before the injection of vehicle or LPS and fivehours later just before the infusion of capsaicin or vehicle. Rectal temperature was

measured 7 hours after the i.p. injection of 37.5 mg/kg LPS, 0.75 mg/kg LPS or vehicle

during the deep anaesthetisation necessary for perfusion. Temperature was not measuredat earlier time-points to prevent interference with the measurement of Fos expression and

behaviour.

BehaviourVideotapes of behaviour shown during the 2 min of infusion and the 10 min. post-

infusion were analyzed with dedicated software (The Observer 3.0, Noldus InformationTechnology b.v., Wageningen, the Netherlands). Because rats had to be moved from the

experimental to the home-cage after the infusion, the behaviour exhibited during the 2

minutes directly after the infusion were not analyzed. The remaining 8 minutes wereanalyzed in 2 periods of 4 minutes (3rd till 7th and 7th till 11th minute after infusion) to

observe if possible initial behavioural differences remained present. Three major types of

behaviour elements were distinguished in the analysis; exploring, immobilization and

discomfort behaviour (table 3.2.1). Occasionally, the animals also showed resting, burying,feeding and scratching/grooming of the body.

Table 3.2.1: Behaviours that were analyzed during intracisternally infusion of vehicle or capsaicin.

Exploring Sniffing and slowly moving around the cage to explore the (new) environment.Occasionally standing on the hind-paws (rearing).

Immobilization All forms of complete immobilization excluding restingDiscomfort Three active behaviours that were interpreted by the investigator as signs of

discomfort that were introduced by the capsaicin infusion into the CM wereincluded in this behaviour:

Head grooming licking of the fore-paws and washing the head.Head scratching licking of the fore -or hind-paws and scratching of the headEscape behaviour rapid moving, turning and rearing, may be jumping, trying to get out

of the cage


81

DrugsThe capsaicin stock solution (3.05 mg capsaicin per 1 ml of saline-ethanol-

Tween80 (8:1:1)) was diluted 1:40 in saline to which 0.2% Evan's Blue (Merck, Darmstadt)

was added. This yields the 250 nM capsaicin concentration. Previous experiments 192

showed that i.c. infused 100 nM capsaicin concentration couldn’t activate the TNC I,IIwhereas 1000 nM i.c. capsaicin gave a maximal activation. To be able to observe LPS

modulation of capsaicin-induced Fos expression in the TNC I,II the intermediate 250 nM

concentration was used in this experiment.LPS (E. Coli Serotype 0.26:B6; Sigma Chemie, Bornem, Belgium) was dissolved in

saline and injected intraperitoneally (i.p.) in concentrations of 0.75 mg/kg LPS (LPS(L)) or

37.5 mg/kg LPS (LPS(H))

Statistical analysesTo elucidate the effects of LPS, statistical analysis was performed separately

amongst the 3 groups of animals that received i.c. vehicle and amongst the 3 groups of

animals that received i.c. capsaicin. The statistical software package Sigmastat ® (Jandel

Scientific, San Rafael) was used to analyze the data. The One Way ANOVA with Student-Newman Keuls test as multiple comparison method (pairwise) was used to test the effects

of different doses of LPS. Sigmastat tests normal distribution and equal variance within the

groups, 2 requirements for the One Way Anova. In cases of non-normal distribution orunequal variance (which occurred occasionally throughout all different behaviours and brain

areas that were counted for Fos positive cells) the non-parametric variant of the One Way

ANOVA, the Kruskall Wallis ANOVA on Ranks with Dunn’s test as multiple comparison(pairwise) was performed. p < 0.05 was considered significant.

Chapter 3.2

82

ResultsLPS; Appearance, Weight-loss and Temperature

Prior to i.c. infusion with capsaicin or vehicle, animals injected with LPS(H) showed

signs of illness including pilo-erection and inactivity. LPS(L) treated animals did not showany signs of illness prior to infusion.

LPS(L) and LPS(H) injected animals both showed significantly more weight-loss

compared to saline injected animals ( 7.0 ± 0.5 g., 6.5 ± 0.6 g. and 4.1 ± 0.6 g. resp., seefigure 2). No significant differences in body temperature could be observed between animals

injected with 37.5 mg/kg LPS, 0.75 mg/kg LPS and saline (37.8 ± 0.2 °C, 37.8 ± 0.2 and

37.5 ± 0.1 °C respectively).

BehaviourThe five behaviours measured (immobilization, exploring, head scratching head

grooming and escape behaviour) accounted for 90.5 percent of all the behaviours shown

during and after i.c. infusion with vehicle or capsaicin. During capsaicin infusion, escapebehaviour was the main discomfort behaviour whereas after infusion, grooming and

scratching of the head were the most shown discomfort behaviours. Primary remaining

Saline LPS(L) LPS(H)0

1

2

3

4

5

6

7*

*

We

ight

los

s (g

r)

Figure 3.2.2: Weight-loss (mean ± S.E.M.) from i.p.injection of either saline, 0.75 mg/kg LPS (LPS(L)) or37.5 mg/kg LPS (LPS(H)) until infusion of vehicle orcapsaicin 5 h. later. * = significantly different fromsaline (p<0.05).


83

behaviours were resting and body grooming which were especially shown by the

LPS(L)+Vehicle and the Control group respectively. Occasionally the animals also showedeating, drinking, burying and scratching of the body.

Exploring behaviourAs is shown in figure 3.2.3, i.c. capsaicin significantly decreases the time animals

spend on exploring behaviour in all 3 periods compared to i.c. vehicle infused animals. The

time spent on exploring behaviour in animals treated with LPS(L)+Vehicle is significantlyreduced in the 3rd till 7th minute after infusion (85.4 ± 15.6 vs. control: 181.6 ± 29.6

seconds) whereas it is reduced during and directly after infusion of vehicle in the

LPS(H)+Vehicle group (52.8 ± 12.0 and 72.4 ± 23.8 vs. Control: 109.6 ± 5.5 and 181.6 ±29.6 seconds respectively). The LPS(H)-induced changes in the time spent on exploring

behaviour have disappeared in the 7th till 11th minute after infusion. Thus differences in

exploring behaviour caused by LPS(H) are transient.The LPS(H)+Caps treated animals display a significant decrease of the time spent

on exploring behaviour both during and 3-7 minutes after the i.c. capsaicin infusion when

compared to the Saline+Caps group. There is no difference in the time spent on exploringbehaviour between the LPS(L)+Caps and the Saline+Caps groups in any of the measured

periods.

ImmobilizationImmobilization behaviour was induced especially in the LPS(H) treated animals.

Animals that were treated with the high concentrations LPS were immobilizing more than 60% of the time during i.c. infusion of either vehicle or capsaicin. In the post-infusion periods

this percentage even increased.

There were no significant differences in the time spent on immobilization behaviourbetween control rats and rats treated with LPS(L)+Vehicle in any of the measured period.

However, during and 3-7 minutes after the i.c. infusion of capsaicin the LPS(L)+Caps group

showed increase of the period spent on immobilization behaviour (46.6 ± 8.1 and 184.8 ±31.6 resp.) compared to Saline+Caps treated animals (24.2 ± 7.2 and 89.9 ± 24.2 resp.).

LPS(L) thus potentiated the time spent on capsaicin-induced immobilization behaviour in

these 2 periods.

DiscomfortSaline+Caps treated animals significantly spend more time on discomfort behaviour (headgrooming, head scratching and escape behaviour) during (20.7 ± 7.7) and 3-7 minutes after

(95.6 ± 30.8) i.c. capsaicin infusion compared to Control rats (during infusion: 0 ± 0, 3-7

minutes after infusion: 9.1 ± 5.7). No differences in time spent on discomfort behaviour are

Chapter 3.2

84

present between LPS(L)+Caps, LPS(H)+Caps and Saline+Caps treated animals. Thus LPScan not modulate the time spent on capsaicin-induced discomfort behaviour.

Min. 1,2 during infusion0

20

40

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Min. 3-6 after infusion Min. 7-10 after infusion0

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240 Control (n=5) LPS(L)+Vehicle (n=5) LPS(H)+Vehicle (n=4) Saline+Caps (n=8)

LPS(L)+Caps (n=7) LPS(H)+Caps (n=8)

A


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34

Immobilization

1

1

2

Tim

e (s

eco

nds

)


40

80

120

160

200

2402B

Figure 3.2.3: Time spent on different kindof behaviours (mean ± S.E.M.) observedduring the 2 minutes of infusion of eithercapsaicin 250 nM (Caps) or vehicle(control) and in two subsequent periods of4 minutes thereafter. Groups LPS(L) andLPS(H) received an i.p. injection with 0.75mg/kg LPS or 37.5 mg/kg LPS respectively5 h. prior to infusion. N.S. means value is 0and therefore not shown. A: Exploringbehaviour. B: Immobilization behaviour. C:Discomfort behaviour. 1, 2, 3 and 4 isrespectively significantly different fromcontrol, LPS(L)+Vehicle, Saline+Caps andLPS(H)+Caps (p<0.05).


85

��

��

TNC I,II0

20

40

60

80

100

120

140

160

180

20034

1

nr. o

f c-f

os p

osi

tive

ce

lls Control (n=5)

�� LPS(L)+Vehicle (n=6)

LPS(H)+Vehicle (n=4) Saline+Caps (n=8)

�� LPS(L)+Caps (n=8)

�� LPS(H)+Caps (n=7)

Figure 3.2.4: Number of c-fos protein positive cells (mean ±S.E.M.) in the outer layers of the trigeminal nucleus caudalis (TNCI,II) of animals treated with i.p. vehicle (control), 0.75 mg/kg LPS(LPS0.75) or 37.5 mg/kg LPS (LPS0.75) injection 5 hours prior tointracisternally infusion of vehicle (control) or capsaicin 250 nM(C250). 1, 2 and 4 is respectively significantly different fromcontrol, LPS0.75 and LPS0.75 + C250 (p<0.05).

Fos expressionTNC

As is shown in figure 3.2.4, LPS does not affect the numbers of cells expressing Fos

protein in the outer layers of the TNC (Vehicle: 10 ± 2, LPS 0,75 mg/kg: 10 ± 1, LPS 37.5

mg/kg: 17 ± 5). The Saline+Caps group however demonstrated an increased number ofFos positive cells in the TNC I,II (103 ± 22) compared to the control group. The

LPS(L)+Caps group showed no differences in TNC I,II Fos expression compared to the

Saline+Caps group. Moreover, although not significant, it was somewhat decreased in theLPS(L) pre-treated animals (61 ± 12). Contrary to the LPS(L)+Caps group, animals treated

with LPS(H)+Caps displayed a significant increase in the number of cells expressing Fos

protein (165 ± 20) in layer I, II of the TNC compared to Saline+Caps treated rats.

NTS and APThe number of Fos positive cells in the AP and the various parts of the NTS is presented intable 3.2.2. The highest (absolute) increase in numbers of cells expressing Fos caused by

both LPS and capsaicin was observed in the caudomedial portion of the NTS adjoining the

AP (Control: 4 ± 2, LPS(L)+Vehicle: 51 ±13, LPS(H)+Vehicle: 152 ± 11, Saline+Caps: 69 ±38). Combined LPS-capsaicin treatment could not significantly alter the Fos expression in

the NTS and the AP compared to combined saline-capsaicin treated animals. C-fos protein

Chapter 3.2

86

expression in the Area Postrema is significantly increased in the LPS(H)+Vehicle andSaline+Caps treated animals when compared to the control group (33 ± 5, 39± 23, 4 ± 3

respectively).

Control LPS(L)+Veh. LPS(H)+Veh. Sal.+Caps LPS(L)+Caps LPS(H)+Caps(n=5) (n=6) (n=5) (n=8) (n=8) (n=7)

mean S.E.M mean S.E.M mean S.E.M mean S.E.M mean S.E.M mean S.E.M

Area Postrema 4 ± 3 14 ± 3 33 ± 512 39 ± 231 40 ± 21 88 ± 34

Caudal NTS Medial 4 ± 2 51 ± 131 152 ± 1112 69 ± 381 80 ± 27 163 ± 46

Caudal NTS Lateral 2 ± 1 7 ± 11 11 ± 212 11 ± 31 14 ± 3 17 ± 5

Rostral NTS Medial 3 ± 1 6 ± 1 12 ± 5 13 ± 4 16 ± 3 25 ± 7

Rostral NTS Lateral 2 ± 0 4 ± 1 4 ± 2 8 ± 21 6 ± 1 5 ± 1

Table 3.2.2: Number of c-fos positive cells in the Area Postrema (AP) and different parts of theNucleus of the Solitary Tract (NTS) in animals treated with i.p. saline (control, sal.), 0.75 mg/kgLPS (LPS(L)) or 37.5 mg/kg LPS (LPS(H)) injection 5 hours prior to intracisternally infusion ofvehicle (control, veh.) or capsaicin 250 nM (Caps.). 1 and 2 is respectively significantly differentfrom control and LPS(L) (p<0.05).


87

Discussion

In the present study, we found that a severe immunological challenge influences

the processing of intracranial trigeminal nociception. This conclusion is based on the finding

that pre-treatment of the animals with 37.5 mg/kg LPS enhanced the number of cells in theTNC I,II that exhibit c-fos protein expression after intracisternally applied capsaicin.

LPS-induced sicknessAn enhanced loss of body weight in animals treated with both the low or high

concentration of LPS was found in the current study. This enhanced weight loss caused by

LPS may be induced by anorexia;99,341,455 or by increased energy consumption associatedwith fever.161,241 As both LPS-induced anorexia and fever seem to be secondary to the

production of cytokines, especially IL-1,161,241,341,456 the weight loss found in the present

study may be considered indicative of a challenged, activated immune system. Although nodifference in enhanced weight loss could be observed between animals treated with the low

or high concentration LPS, other signs of an activated immune system do indicate

differences in sickness severity between LPS(L) and LPS(H) treated animals. The pilo-erection and loss of posture shown by LPS(H) treated animals were not quantified but

reduction of locomotor activity, which is a well known sickness behaviour after LPS

treatment,341,494 was quantified by measuring the time of immobilization shown by LPStreated rats during the infusion of vehicle solution. As capsaicin itself also induces

immobilization behaviour, effects of LPS on immobilization behaviour can only be studied in

animals that are not subject to capsaicin infusion. Control rats almost exclusively showexploring behaviour during the two minutes of vehicle infusion. The novel environment of

the cage that is used during infusion most likely induces this behaviour. During i.c. infusion

of vehicle only the LPS(H) concentration induced immobilization behaviour and a significantreduction of exploration. This indicates that although the weight loss in the LPS(L) and

LPS(H) treated animals is comparable, the LPS(H) treated animals do suffer more from the

higher LPS concentration compared to the LPS(L) treated animals.The concentrations of LPS-type E.Coli 0.26:B6 used in the present experiments

seem relatively high compared to other studies using LPS.161,241,341 However the lack of

changes in immobilization behaviour and the absence of pilo-erection in the LPS(L) treatedanimals shows that relatively high concentrations of this LPS-type are necessary to induce

sickness behaviour.

LPS sensitization of capsaicin sensitive afferentsThere are two findings in this study that point to LPS potentiation of capsaicin-

induced intracranial trigeminal activation. First of all there is the above-mentionedenhancement of capsaicin-induced Fos expression in the outer layers of the TNC by LPS(H).

Chapter 3.2

88

The TNC is the primary relay station for nociceptive trigeminal afferents150 and especiallycells in layer I and II of the TNC are termination sites of small unmyelinated C-fibers that

react to nociceptive stimuli.187,322 As Fos is considered a marker for sensory neuronal

activation,162 the increased number of cells that express c-fos protein in the outer layers ofthe TNC indicates (1) that more nociceptive trigeminal afferents become activated or (2)

that the synaptic transmission from first to second order neurons in the TNC is enhanced.

Thus LPS enhances nociceptive trigeminal processing.A second finding from this study that points to potentiation of capsaicin-induced

effects by LPS is that although LPS(L) alone did not induce immobilization behaviour, this

low dose could enhance the capsaicin-induced immobilization behaviour. This effect ofLPS(L) on immobilization behaviour does suggest a sensitization of intracranial trigeminal

afferents.

An important issue in considering the effect of LPS(L) on capsaicin-inducedimmobilization behaviour is that LPS(L) could not potentiate capsaicin-induced Fos

expression in the TNC I,II. Moreover, Fos data point to hyposensitivity rather than

hypersensitivity of the trigeminal afferents. This discrepancy between TNC I,II Fosexpression and immobilization behaviour may be explained by the difference in temporal

resolution of both parameters. As Fos is an accumulation of cell activating events in the

hours preceding perfusion, Fos in the TNC I,II has little to no temporal resolution if it isconsidered as parameter of trigeminal nociception. Immobilization behaviour however is

measured acutely during the i.c. infusion of capsaicin and directly hereafter. The increase in

capsaicin-induced immobilization behaviour by LPS(L) is transient and may therefore not bereflected in TNC I,II Fos expression.

An alternative explanation for the discrepancy between Fos expression in the TNC

I,II and immobilization behaviour is that capsaicin may activate not only trigeminalpathways but also other afferent pathways. Two alternative pathways may especially be

relevant in this model.

Capsaicin is infused into the Cisterna Magna. This site is located near the AreaPostrema, a brain region that contains capsaicin receptors.428 The Area Postrema projects

heavily to the NTS,101 which is the primary relay station for visceral afferent information and

the NTS has pronounced ascending projection patterns throughout the brain.437 Capsaicinenhanced the Fos expression of both the AP and caudomedial NTS, confirming that this

pathway is activated.

A second alternative pathway, also involving the NTS, that might be activated afterintracisternal capsaicin infusion is mediated through vagal afferents. Vagal afferents are a

possible target for intracisternally applied capsaicin because they innervate both the basilar

artery and the dura mater.190,191 Several branches of vagal afferents are sensitive forcapsaicin155,247 and vagal afferents can contain Substance P (SP),114 a neuropeptide that,


89

although debated in migraine85,136 has been associated with nociception of the

head.107,309,314

Also, like trigeminal afferents,151,424 specific vagal afferent branches49 and ganglia80

have been reported to show CGRP immunoreactivity and CGRP mRNA respectively. CGRP in

the venous outflow of the head has been found elevated in migraine patients during amigraine attack128 and in nitroglycerin-induced cluster headache,100 associating this

neuropeptide with headache. Additionally the increased release of CGRP from rat tracheal

perfusates after nociceptive treatment with capsaicin as described by Hua and colleagues,161

is thought to be derived from vagal afferents that innervate the trachea.

Thus vagal afferents, like trigeminal afferents, are 1) localized in the meninges 2)

contain neuropeptides that are associated with headache and 3) are reactive to capsaicin.As the primary termination site of vagal afferents in the brain is the NTS, counting the

number of Fos positive cells in the NTS, as was done in these experiments, could possibly

elucidate whether capsaicin-induced activation of the NTS can be enhanced by LPS(L). Thisin turn might explain that LPS(L) enhances the capsaicin-induced immobilization behaviour

without affecting the Fos expression in the TNC I,II. Capsaicin indeed does induce Fos

expression in the NTS, especially in the caudomedial part that receives input from generalvisceral afferents (opposite to the rostral part that predominantly receives gustatory

information-reviewed by Saper368). However, as LPS(L) alone also induces Fos expression in

that part of the NTS, a potential enhancement of capsaicin-induced Fos expression in theNTS by LPS(L) can not be detected.

LPS-induced hyperalgesiaSeveral explanations for increased sensitivity of nociceptive afferents after an

immunological challenge have been put forward in literature. A neurocircuitry has been

proposed to be involved in illness-induced hyperalgesia.475 The tail flick latency to radiantheat was tested in several conditions. Using this paradigm it was demonstrated that illness

inducing agents produce hyperalgesia by initiating the production of cytokines. The

cytokines in turn activate a pathway that subsequently involves the hepatic branch of thevagus, a circuitry in the brain involving the NTS and probably the nucleus raphe magnus

and dorsal medial hypothalamus and a pathway in the dorsal funiculus of the spinal cord.475

Although hyperalgesia was tested 1 hr. after LPS administration instead of 5 h. used in thepresent investigation, evidence for involvement of this neurocircuitry in our experiments is

found in the dose dependent increase of Fos expression in the NTS after LPS administration.

Besides affecting hepatic vagal nerves, cytokines produced after LPS administrationalso act directly within the brain. Several cytokines, including TNF-α and IL-1β are

transported from blood to the brain by a saturable transport system.18

Intracerebroventricular (i.c.v.) injection of IL-1β is able to induce anorexia343,408,455,456 in

rats. Also, in two different animal models225,282 anorexia-induced by a peripheral

Chapter 3.2

90

immunological challenge could be antagonized by central administration of an interleukin 1receptor antagonist. The enhanced decrease in body weight induced by LPS treatments in

the present study may indicate involvement of central IL-1 receptors. Interestingly,intracerebroventricular injection of IL-1β has been shown to induce hyperalgesia at the level

of the trigeminal nucleus after stimulation of extracranial trigeminal afferents in rats.326 Theneuronal substrate for this central action of IL-1β probably resides in the hypothalamus.372

Thus, cytokines might induce hyperalgesia in the present experiments by acting atperipheral vagal afferents or at central brain regions. A final site of action of cytokines that

has to be considered is the effect that cytokines may have at the intracranial nociceptiveafferents themselves. LPS, and more specific the cytokines TNF-α and IL-1β are able to

increase the capsaicin-induced CGRP release from tracheal afferents.161 The immunological

stimulation was performed in vivo but as the capsaicin stimulation was performed in in vitroexperiments (tracheal perfusates that were dissected from rats), it can be excluded thatspecific brain regions are involved in facilitating the capsaicin-induced CGRP release. Morelikely, the cytokines TNF-α and IL-1β act directly at the afferent nerve terminals.

Concluding, cytokines that are produced after LPS infusion might act at hepaticvagal afferents, central brain regions or nociceptive afferent nerve terminals to induce

hyperalgesia. These different actions of cytokines do not exclude each other. Experiments

are currently performed to elucidate which mechanism is most relevant for the hyperalgesiceffects of LPS on intracranial trigeminal afferents.

Possible role of immune system-induced hyperalgesia in headache and migraineThe most pronounced effect found in this study is that LPS(H) could potentiate the

capsaicin-induced TNC I,II Fos expression. Although the 37.5 mg/kg LPS concentration is

quite severe it strongly suggests that trigeminal afferents can become hyperalgesic after animmunological challenge. This may explain the reports that migraineous and non-

migraineous headache are of highest intensity after infection.55

Evidence in literature supports the relationship between infections and headache.Not only head and neck infections cause headache496 but also well known infections like

influenza and HIV are linked to headache.84,318 Less well known infections like Japanese

spotted fever and intrasellar infection are also associated by headache.31,252 Well describedis the headache after encephalitis, sinusitis or meningitis,42,360,375,469 all infections of the

head. It is clear from the above that different kind of infections can trigger headache. The

present results suggest that the association between infections and headache may involveimmune activation-induced hyperalgesia of nociceptive sensory nerves of the head.

Several reports of altered immune system function in migraine patients have beenput forward by Covelli and colleagues.66,68-70 Increased spontaneous TNF-α release and a

deficit of killing / phagocytosis of polymorphs and monocytes was found in migraine patients

without aura compared to controls66,68,70. Also a significant increase in T-lymphocyte subsets


91

was found in migraine patients compared to healthy controls.270 It is suggested that these

altered immune system functions may be responsible for the vascular, haemodynamic andpro inflammatory symptoms associated with migraine.69 However no differences in TNF-αand IL-1 plasma levels could be found during and in-between attacks458 in migraine patients

suggesting that these cytokines are not involved in the initiation of the attack. Nevertheless,immunological dysfunction could make migraine patients more susceptible to migraine

initiating events by causing hyperalgesia of nociceptive afferents of the head. This is

supported by the present results.Concluding: The number of cells expressing Fos in the outer layers of the TNC after

capsaicin treatment is increased by 37.5 mg/kg LPS. Thus trigeminal nociceptive processing

can be enhanced by a severe immunological challenge. Immobilization behaviour, inducedby intracisternally applied capsaicin, can be enhanced by 0.75 mg/kg LPS. These

hyperalgesic effects of LPS on capsaicin sensitive afferents are probably mediated by

cytokines that act at peripheral vagal nerves, central brain regions or via direct actions ofcytokines on capsaicin sensitive afferent nerve terminals. The found hyperalgesic action of

LPS on trigeminal and possibly other capsaicin sensitive afferents of the head may explain

the reports that headache can be triggered by different types of infections and thatmigraineous and non-migraineous headache is of highest intensity after an infection.

Chapter 3.2

92

Section 4

Central pharmacologicalmodulation of trigeminovascular

headache

Section 4

94

PREFACE

Many anti-migraine drugs with possible peripheral and central target sites were already

tested in models of trigeminovascular nociception using anaesthetized animals. Theadvantage of the model presented in this thesis is the ability to study trigeminovascular

nociception induced behaviour as derivative of cumulative cerebral activity. Therefore, the

effects of two drugs that may inhibit trigeminovascular pain processing by centralmechanisms will be presented in this section.

The first chapter describes the results with the long acting somatostatin analogueoctreotide. Octreotide has already been successfully tested in migraine but it poorly

penetrates the brain. An extensive system of somatostatin positive fibers and receptors is

present in the TNC I,II, which is considered as a possible target for the modulation oftrigeminovascular pain processing. The efficacy of centrally applied octreotide was tested

using intracisternal injections 10 minutes before the application of capsaicin.

The second chapter describes our findings with the neuronal nitric oxide syntase (nNOS)

inhibitor 7-NitroIndazole (7-NI). NO is considered a key molecule in the pathophysiology of

migraine and 7-NI has shown anti-nociceptive activity in several pain models. As 7-NI easilydiffuses into tissue, it was applied intraperitoneally 30 minutes before the activation of the

trigeminovascular system.

Octreotide and trigeminovascular nociception

95

Chapter 4.1

Intracisternally applied octreotide does not ameliorate orthodromictrigeminovascular nociception1

SummaryOctreotide is a somatostatin analogue that has been effectively used to treat

migraine. Octreotide poorly penetrates the blood-brain barrier, but has potential centraltarget sites in the trigeminal nucleus caudalis, which is the primary central relay station for

trigeminal nociceptive information in the brain. We studied the effect of intracisternally-

applied octreotide in a model of trigeminovascular stimulation in the unrestrained rat usingintracisternal capsaicin infusion to stimulate intracranial trigeminal nerves. Fos expression in

the outer layers of the trigeminal nucleus caudalis (TNC I,II) and behavioural analysis was

used to measure the effects of octreotide on capsaicin-induced trigeminovascular activation.Octreotide-induced head grooming and scratching behaviour indicating an effect of

octreotide on the trigeminovascular system. However, octreotide did not alter the average

capsaicin-induced Fos expression in the TNC I,II and capsaicin sensitive behaviours werealso not modified by octreotide pre-treatment. This argues against a role for central (TNC

I,II) somatostatin receptors in the processing of trigeminovascular nociception.

1 with: M. Jeuring, W.J. Meijler, J. Korf and G.J. Ter Horst. Submitted forpublication in Cephalalgia

Chapter 4.1

96

IntroductionSomatostatin (somatotropin release-inhibiting factor (SRIF)) is a hypothalamic

pituitary regulatory hormone, which is present in brain, spinal cord,271 gut and pancreas.355

It suppresses growth hormone release and inhibits the release of regulatory peptides of thegastroenteropancreatic endocrine system.137,355 Octreotide (SMS 201-995) is a long-acting

somatostatin analogue, which, due to its inhibitory actions on growth hormone and gastric

peptide release, is used in patients suffering from acromegaly, cancer, gastrointestinaldiseases and pancreatitis (see21). Until now, 5 somatostatin receptor genes have been

cloned (sst1 to sst5) that fit within previously defined SRIF1 and SRIF2 groups. The SRIF1

group consists of the sst2, sst3 and sst5 receptor with relative high (sst2, sst5) to moderate(sst3) affinity for octreotide and a marked structural similarity. The SRIF2 group consists of

the sst1 and sst4 receptor with low affinity for octreotide and also a high mutual structural

similarities.160 The sst2 receptor has been identified in two spliced isoforms, the sst2(a) andsst2(b) receptor that have similar binding properties but may differ in G-protein

coupling.459,460

Somatostatin and octreotide have effectively been used to relief clusterheadache394 and migraine182 respectively. The target of action of octreotide for migraine

relief is not known but is most likely peripheral as it can poorly penetrate the brain.197,378 Its

mode of action may involve inhibition of release of several vasoactive substances sincesomatostatin can inhibit trigeminal substance P release,41 a neuropeptide that has been

implicated in the pathophysiology of migraine.78,309,313,314,319 Antidromic release of substance

P from sensory nerve endings, and the vasodilatation it triggers, are inhibited bysomatostatin administration.118 Octreotide has, like many other anti-migraine

drugs,45,48,262,365 effectively been used to antagonise neurogenic plasma protein

extravasation in the dura mater after trigeminal ganglion stimulation and intravenouscapsaicin administration.275 As octreotide could not reduce (not neurogenic) substance P-

induced plasma protein extravasation, the mode of action of octreotide most likely involves

receptors located at the prejunctional trigeminal sensory nerve endings that innervate thedura mater.275

An extensive complex of somatostatin containing neurons and fibers is present in

the rat trigeminal nucleus caudalis, layer I,II (TNC I,II), the primary relay nucleus fortrigeminal nociceptive signals.3,4,492 Somatostatin positive fibers in layer II originate

predominantly from primary trigeminal afferents, whereas somatostatin immunoreactivity in

layer I most likely originates from interneurons in layer I and II of the TNC.3,4 Receptor sitesfor somatostatin, based on autoradiography with the (Tyr3) derivative of octreotide,

(125I)204-090, accordingly have been reported in the substantia gelatinosa of the trigeminal

nuclear complex of rat357 and human.356 Moderate densities of sst3 receptor mRNA339,384 andsst2(b) receptor mRNA377 have been shown in the spinal trigeminal nucleus. This provides a

potential central target for octreotide to modulate orthodromic trigeminal nociception.


97

Centrally acting octreotide-like drugs may show improved analgesic efficacy in migraine due

to inhibition of both antidromic and orthodromic trigeminovascular pain processing.Aim of this study was to evaluate the effects of octreotide in a conscious rat model

of intracranial trigeminovascular nociception. Because octreotide poorly penetrates the blood

brain barrier and octapeptide analogs of somatostatin,431 somatostatin itself, DC 32-87 (sst2selective agonist) and BIM-23056 (sst3 selective agonist)117,142 showed modulation of

physiological effects mediated by regions in the brainstem after intracisternal infusion, we

choose this route of drug administration.The larger blood vessels of the brain and the meninges are innervated by sensory

nerves of the trigeminal system, together forming the trigeminovascular system. Several

animal models of trigeminovascular activation have shown to be predictive for analgesiceffects of anti-migraine drugs,45,48,75,158,262,275,322,365 including the newly developed triptans

with a central site of action.64,129 A previously described animal model of intracranial

trigeminovascular stimulation, based on the intracisternal infusion of the irritant capsaicin inconscious rats was used since it enables the analysis of behaviour combined with

assessment of Fos immunoreactivity in the trigeminal nucleus caudalis.192

Chapter 4.1

98

MethodsThe experiments were performed according to the ethical guidelines for

investigations of experimental pain in conscious animals503 and were approved by the local

committee of Animal Bio-Ethics, University Groningen (FDC 2198). Male Wistar ratsweighting 250 – 325 gr. were used. They were individually housed in a light/dark cycle with

lights on from 08:00 till 20:00 hour. Food and water were provided ad libitum.

Cisterna magna (CM) cannulationThe cannula was made from a stainless steel needle (0.6 x 25 mm. 23 G x 1’’;

Braun, Melsungen, Germany) of which 6.5 mm was inserted into the brain. Surgery wasperformed under semi-sterile conditions. Sodium pentobarbital was used as anaesthetic (60

mg/kg, i.p.). Rats were placed in a stereotaxic apparatus with incisor bar at –7 mm from the

horizontal plane. Two holes were drilled into the caudal corners of the interparietal skull andtwo screws (diameter 1.0 mm) were driven 1.5 mm into the skull. A hole of 1.2 mm was

drilled at the midline of the external occipital crest through which the CM cannula was

carefully placed behind the cerebellum, into the CM. Correct placement was confirmed bywithdrawal of cerebrospinal fluid. The cannula was fixed to the skull with dental cement

(Kemdemt, Purton Swindon, UK) and closed by insertion of a metal wire (of the same length

as the cannula) within a polyethylene cap to seal the cannula off.

DrugsCapsaicin (3.05 gr.) was dissolved in 1 ml saline-ethanol-Tween 80 (8:1:1) (vehicle-

stock) and sonicated for 5 minutes (capsaicin-stock). The capsaicin-stock and vehicle-stock

solutions were further diluted 1:40 to yield the capsaicin 250 nM and vehicle solution

respectively. Evans Blue (0.2% EB) was added to both the solutions to determine thedistribution of infused solution after the experiments. Octreotide was provided asSandostatin ® in a concentration of 5 µg per 10 µl. Saline was used as control solution for

the octreotide solution.

Experimental procedures.Octreotide or saline was injected intracisternally through the cannula in a volume of

10 µl, 10 minutes prior to infusion of capsaicin or vehicle. During the injection we attached a

silica tube with internal diameter of 75 µm to the microinjector to reduce the internal

diameter of the CM cannula.For the infusion of 100 µl capsaicin or vehicle the rats were placed in an observation

cage (30x30x30 cm). This amount was infused in 2 minutes by a microinjectorpump

(CMA100, Carnegie Medicin, Stockholm, Sweden). The behaviour of rats was recorded onvideotape from 5 minutes before to 10 minutes after the infusion of capsaicin or vehicle.


99

Perfusion and immunohistochemistryRats were perfused 2 h. after the infusion of capsaicin or vehicle. Prior to the

transcardial perfusion rats were deeply anaesthetized with sodium pentobarbital (120 mg/kg

i.p.) and perfused with 0.9% saline for 1 min, followed by 4% paraformaldehyde (PF) in 0.1

M phosphate-buffered saline (pH 7.4) for 20 min. After removal of the occipital bone,placement of the cannula in the CM was confirmed and the distribution of infused solution

was determined by inspection of the Evans Blue staining pattern. After the removal, the

brains were post-fixed in 4% PF during 24 h. Prior to sectioning the brain was cryoprotectedby overnight storage in 30% sucrose in 0.1 M phosphate buffer (pH 7.4). Forty µm thick

coronal serial sections were prepared on a cryostat microtome at -15°C, and collected in 0.2

M potassiumphosphate-buffered saline (KPBS, pH 7.4) with sodiumazide (0.1%).

Free floating sections were immunocytochemically stained for the proto-oncogene

protein c-fos according to a standard protocol previously described.192,193 In brief, afterpreincubation in normal sera and pre-treatment with 0.3 % H2O2, the primary antibody,

rabbit anti-Fos (1:10000; AB5, Oncogene Science inc, Cambridge, UK) was applied overnightat room temperature. After washing biotinylated goat-α-rabbit IgG(1:200, Pierce, Rockford)

was applied for 2 hours. Subsequently, the sections were washed and then incubated with

the avidine-biotine-peroxidase complex (Vector Labs, Burlingame) for 2 h. at room

temperature. The Ni-enhanced 3,3’-diaminobenzidine tetrahydrochloride reaction was usedto visualize the presence of peroxidase. Intermittent washing was performed with KPBS,

antibodies were dissolved in KPBS with 0.5% triton X-100. All staining procedures were

performed with gentle agitation. Sections were mounted, dehydrated and coverslipped withDEPEX.

QuantificationFos expression

Fos-ir cells in layer I and II of the TNC (TNC I,II) were counted at -1, -2, -3, -4, -5

and -6 mm caudal from obex by an observer blinded from experimental procedures.Sections from -0.5 to -1.5 mm were averaged to obtain the count for the -1 mm level and

so on. The mean of the total TNC I,II was calculated by averaging the Fos expression at the

6 levels.

BehaviourThe behaviour shown before, during and after the infusion was analysed with

Observer ® software (Noldus Information Technology, Wageningen, the Netherlands). The

first 2 minutes immediately after infusion were not analysed because rats were uncoupled

from the microinjector device in that period. Behaviours scored were exploring behaviour(exploring and rearing), head grooming and head scratching and immobilization (all forms

Chapter 4.1

100

other than resting). Other behaviours shown include body grooming, eating and drinking,and resting.

Satistical analysisThe following groups were assembled: Saline + vehicle (Control, n=3), octreotide

+ vehicle (Oct, n=4), Saline + capsaicin (Caps, n=5) and octreotide + capsaicin (Oct+Caps,

n=7). Effects of octreotide independent of capsaicin were tested by comparing the Oct andthe Control group. Effects of octreotide on capsaicin-induced numbers of Fos positive cells in

the TNC I,II and behaviour were tested by comparing the Oct+Caps group to the Caps

group. The Student’s t-test was used to test significant differences if data showed normaldistribution, in all other cases the Mann-Whitney Rank Sum test was employed. P < 0.05

was considered significant. Fos data are expressed as mean number of positive cells ±

S.E.M. and behavioural data as mean time in seconds spent on each behaviour ± S.E.M.

ResultsInclusion criteria

As previously described,192 only rats with successful infusions exhibiting dural EB

staining patterns ventral from the hindbrain, around the brainstem and the upper levels ofthe cervical spinal cord were included.

BehaviourPrior to noxioustrigeminovascular stimulation(fig. 4.1.1)

Behavioural data

obtained from either

octreotide or saline treatedrats in the 5 minutes prior to

the infusion of capsaicin or

vehicle were pooled regardlessof the subsequent type of

infusion. Octreotide caused a

significant decrease ofexploring behaviour (saline:

210 ± 12, octreotide: 141 ±

20) and a significant increaseof head grooming / scratching

(saline: 34 ± 9, octreotide:

Exploring Immobilisation Head grooming/scratching0

25

50

75

100

125

150

175

200

225

250 Saline Octreotide

**

Tim

e (s

ec)

Fig 4.1.1. Effects of octreotide on the time spent ondifferent kind of behaviours (mean ± SEM) observedduring the 5 minutes prior to intracisternal capsaicin orvehicle infusion. Saline: n=8. Octreotide: n=11. * issignificantly different from saline treated animals (p <0.05).


101

117 ± 22). Changes of immobilization behaviour (saline: 20 ± 8, octreotide: 10 ± 5) or body

grooming / scratching (saline: 13 ± 5, octreotide: 8 ± 3) were not observed.

During noxious trigeminovascularstimulation (fig. 4.1.2)

Octreotide-induced

modulation of exploring, head

grooming and head scratchingbehaviour was not evident during

the 2 minutes of infusion of

vehicle (exploring: Control: 106± 3, Oct: 105 ± 6; head

grooming / scratching: Control:

10 ± 2, Oct: 8 ± 7). Also,intracisternal infusion of

octreotide could not prevent the

capsaicin-induced reduction ofexploring behaviour induced by

capsaicin (Caps: 27 ± 7,

Oct+Caps: 32 ± 4). Also, theinduction of head grooming and

head scratching in capsaicin

treated animals (Caps: 70 ± 9,Oct+Caps: 65 ± 8) was not

prevented by octreotide.

Capsaicin, octreotide, or thecombination of these 2

compounds did not initiate

alterations in immobilizationbehaviour compared to control

animals.

After noxious trigeminovascularstimulation (fig. 4.1.3)

Octreotide followingvehicle infusion caused a

significant reduction of the

exploring behaviour (Control: 333 ± 14, Oct: 194 ± 51) but head grooming, head scratching(Control: 62 ± 24, Oct: 132 ± 71) and immobilization behaviour (Control: 47 ± 21, Oct: 121


10

20

30

40

50

60

70

80

90

100

110

120 Control Oct Caps Oct+Caps

*

*T

ime (

sec)

Fig 4.1.2. Effects of intracisternally applied octreotide oncapsaicin and vehicle induced behaviours (mean ± SEM)observed during the 2 minutes of infusion. Various groupsare treated with saline + vehicle (Control, n=3), octreotide+ vehicle (Oct, n=4), Saline + capsaicin (Caps, n=5) andoctreotide + capsaicin (Oct+Caps, n=7) treated animals. *is significantly different from Control (p < 0.05).

Fig 4.1.3. Effects of octreotide on the time spend ondifferent kind of behaviours (mean ± SEM) observedduring 8 minutes after infusion of capsaicin or vehicle.Various groups are treated with saline + vehicle (Control,n=3), octreotide + vehicle (Oct, n=4), Saline + capsaicin(Caps, n=5) and octreotide + capsaicin (Oct+Caps, n=7).N.S. means value is 0 and therefore not shown. * issignificantly different from Control (p < 0.05).


50

100

150

200

250

300

350

400

450

*

Control Oct Caps Oct+Caps

NSNS

*

*

Tim

e (

sec)

Chapter 4.1

102

± 63) were not significantly altered by this treatment. Octreotide pre-treated animals werenot significantly different from saline pre-treated animals in their reaction to capsaicin. Time

spent on immobilization (Caps: 395 ± 25, Oct+Caps: 313 ± 36), head grooming and head

scratching behaviour (Caps: 79 ± 24, Oct+Caps: 161 ± 35) was not altered by thetreatment. Moreover, there was a trend of increased head grooming and head scratching in

the octreotide pre-treated rats.

Fos expression(fig. 4.1.4)Intracisternally

applied octreotide alone doesnot significantly alter Fos

expression in the TNC I,II,

although it was increased at alllevels of the TNC (Average:

Control: 72 ± 7, Oct: 145 ±

42). Capsaicin induced amarked increase of Fos

expression at every rostro-

caudal level of the TNC I,II(Average: Caps: 643 ± 50).

Octreotide did not significantly

alter the average Fosexpression in the TNC I,II

(Oct+Caps: 610 ± 15) but

caused a small, significantdecrease of the number of capsaicin-induced Fos positive cells at 6 mm caudal from obex

(Caps: 800 ± 44, Oct+Caps: 702± 23).

Fig 4.1.4. Number of Fos positive cells (mean ± SEM) inlayer I and II at several levels of the trigeminal nucleuscaudalis in animals treated with: saline + vehicle (Control,n=3), octreotide + vehicle (Oct, n=4), saline + capsaicin(Caps, n=5) and octreotide + capsaicin (Oct+Caps, n=7). *is significantly different from Control, # is significantlydifferent from Caps (p < 0.05).

1 2 3 4 5 6 Average0

100

200

300

400

500

600

700

800

900

#

*

**

*

**

*

Nr.

of F

os

posi

tive c

ells

Distance caudal to obex (mm)

Control Oct Caps Oct+Caps


103

DiscussionOctreotide infusions into the cisterna magna reduced the exploring behaviour and

increased the head grooming / scratching behaviour. Intracisternal octreotide pre-treatment

did not affect the capsaicin sensitive behaviours and accordingly did not reduce the average

number of capsaicin-induced Fos positive cells in the TNC I,II. A small but significantreduction of capsaicin-induced Fos expression by octreotide was found in the caudal-most

part of the TNC I,II.

That octreotide affects the trigeminal system intracranially can be concluded fromthe head grooming/scratching behaviour that was observed before the infusion of capsaicin.

In control animals, the proportion head grooming to head scratching is 6.5. In octreotide

treated animals, both behaviours are increased but the proportion head grooming to headscratching is 1.5, showing that especially the head scratching is increased in octreotide

treated animals. In former experiments,192 we observed especially head scratching at higher

concentrations of capsaicin, indicating that octreotide in these experiments may act as anirritant. This is confirmed by reports that describe pain at the (subcutaneous) injection site

of octreotide in humans (see21). There was no induction of body grooming or scratching in

the rats (close to zero in all groups) so the effect seems restricted to the afferents of thehead, rather than being a centrally mediated general effect on behaviour. This is however

not confirmed by the Fos expression in the outer layers of the TNC, the primary relay station

for trigeminal nociceptive afferents. Although the Fos expression at every level of the TNCI,II of octreotide treated animals is higher compared to control animals, the difference did

never reach statistical significance. That the changes in behaviour caused by octreotide are

not well reflected in TNC I,II Fos expression may be caused by the difference in temporalresolution of both parameters. Behaviour is measured acutely, whereas Fos expression is an

accumulation of cell activating events in the hours preceding perfusion. Also, as was shown

by Bereiter and colleagues, some corneal responsive neurones in the TNC do not expressFos after trigeminal (corneal) stimulation while electrophysiological experiments do show

that they are activated.30

Despite the above mentioned actions of octreotide in the trigeminovascular system,intracisternal octreotide was not effective in reducing the average capsaicin-induced

expression of Fos in the TNC I,II. Furthermore, the behaviours sensitive for capsaicin

treatment were not affected by octreotide pre-treatment indicating that secondary or higherorder trigeminal processing is not modified by intracisternal octreotide administration. These

results are confirmed by Bereiter and colleagues who observed that the Fos expression in

the largest part of the TNC after stimulation of the cornea was not modified by i.c.voctreotide pre-treatment. A reduction of Fos expression by octreotide was only seen in the

caudal-most part of the TNC after trigeminal corneal stimulation, which may implicate

different sensitivity for octreotide of trigeminal neurons along the rostro-caudal axis.30 It hasto be noted that in our experiments also a small but significant reduction was found in the

Chapter 4.1

104

caudal-most part of the TNC. In terms of inhibiting orthodromic acute central trigeminal painprocessing for treatment of trigeminovascular headaches like migraine, this is likely not

relevant.

When stimulated, trigeminovascular afferents in the dura mater releaseneuropeptides antidromically, that causes the process of plasma protein extravasation (PPE)

in the dura mater. This process, initiated by antidromic conduction in the trigeminovascular

system can be inhibited by octreotide. When administered intravenously in guinea pigs orrats, i.v. octreotide inhibits dural PPE, elicited by capsaicin or trigeminal ganglion

stimulation.275 This effect on antidromic release of neuropeptides in the trigeminovascular

system may be the reason why migraineurs benefit from subcutaneous octreotidetreatment.182

Due to the direct stimulating effect of intracisternally applied capsaicin on

intracranial trigeminal afferents, the acute behavioural effects and induction of Fosexpression in the TNC I,II are a measure of orthodromic activity, rather than antidromic

activity in the trigeminovascular system. The presence of somatostatin fibers, receptors3,4,357

and octreotide receptor mRNA in the trigeminal nucleus,339,377,384 indicated that centrallyapplied octreotide may modulate the orthodromic processing of trigeminovascular

nociception. However, our experiments demonstrated that the orthodromic conduction in

the TNC I,II is not modified by octreotide administration, at least not to such extent that itmay be an additional target for treating the pain of trigeminovascular headaches like

migraine.

The difference in efficacy of octreotide at orthodromic or antidromictrigeminovascular nociceptive processing, may point to the relatively different roles of

neuropeptides at the afferent nerve terminals centrally or in the peripheral vascular tissue.

At the central terminals of trigeminal afferents, neuropeptides like substance P andsomatostatin most likely act as modulators of the classical neurotransmitters, like glutamate.

The outer layers of the dorsal horn are predominantly innervated by terminals containing

glutamate and co-localization with substance P and CGRP has been shown in theseregions.289 Glutamate is also involved in the processing of pain at the level of the TNC I,II105

and endogenous pain control in the trigeminal nucleus by descending noradrenergic and

serotonergic systems acts possibly through the inhibition of glutamate release.449 In fact,recently it has been shown that NMDA receptor blockade with MK-801 does attenuate the

Fos expression in the TNC I,II after intracisternal capsaicin treatment296 and reduces the

activity in the TNC following electrical stimulation of the trigeminal ganglion.61 This arguesfor an important role for glutamate in the processing of trigeminovascular nociception at the

level of the TNC I,II. At the peripheral perivascular terminal sites however, neuropeptides

act as the primary transmitter, which are able to cause PPE. Glutamate is not a likelycandidate for peripheral release as NMDA receptor blockade, for example, does not inhibit

PPE induced by sciatic nerve stimulation.180 Therefore, modulation of neuropeptide release


105

in trigeminal afferents, may bring about larger effects on the induction of PPE

antidromically, than on the transmission of nociceptive signals orthodromically.

Chapter 4.1

106

7-NI and trigeminovascular nociception

107

Chapter 4.2

Neuronal nitric oxide synthase inhibition in acute trigeminovascular nociception1

SummaryThe nitric oxide (NO) donor nitroglycerin is able to induce migraineous headache in

migraineurs, and NO is thought to be a key molecule in the development of

trigeminovascular headaches like migraine in general. Neuronal NO synthase (nNOS)inhibition by 7-nitroindazole (7-NI) has shown anti-nociceptive activity in some animal pain

models. Aim of this report was to study the role of nNOS derived NO in a conscious animal

model of acute trigeminovascular nociception. Intracisternal infusion of capsaicin was usedto stimulate the trigeminovascular system and treatment with 7-NI (50 mg/kg i.p., 30

minutes prior to trigeminovascular stimulation) was used to inhibit the production of NO by

nNOS. Fos immunoreactivity (Fos-ir) in the trigeminal nucleus caudalis, layer I,II (TNC I,II)was used to assess activity of the nociceptive part of the trigeminovascular system. As there

are multiple targets for 7-NI inside the brain, no anaesthetics were used, so behaviour could

be analysed. The behavioural results prior to the infusion of capsaicin or vehicle show that7-NI increases immobilization behaviour and reduced head grooming / scratching behaviour

compared to control animals. During infusion, capsaicin caused a significant decrease of

exploring behaviour and a significant increase of immobilization and head grooming /scratching behaviour compared to control animals but none of these capsaicin-modified

behaviours were altered by 7-NI pre-treatment. In concordance, the capsaicin-induced Fos-

ir in the TNC I,II was not significantly altered by 7-NI. These results do provide evidenceagainst a role for neuronal derived NO in acute trigeminovascular nociception.

1with: M.B. Spoelstra, G. Vogt, W.J. Meijler, J. Korf and G.J. Ter Horst.

Chapter 4.2

108

Introduction

Migraine affects approximately 10% of the human population. The

pathophysiological mechanisms underlying migraine are still unclear but there is generalagreement that the headache part of the migraine attack originates from the complex of

bloodvessels in the meninges that are innervated by trigeminal nociceptive

afferents.103,125,221,311,373 One of the key molecules, thought to be associated withnociception in the trigeminovascular system is nitric oxide (NO). The NO donor nitroglycerin

can induce a migraine attack in migraineurs and headache in non-

migraineurs,167,169,221,329,442 and nitroglycerin-induced headache can be antagonized by theanti-migraine drug sumatriptan168 Plasma histamine levels, and the spontaneous release of

histamine from leukocytes are increased in migraineurs138,146,367,382,383 and it has been

demonstrated that histamine causes migraine in migraineurs through NO-dependentmechanisms.221

The production of NO from L-arginine is catalysed by the enzyme nitric oxide

synthase (NOS) and NO can be derived from endothelial NOS (eNOS), neuronal NOS(nNOS), or from macrophages and astroytes (inducible NOS, iNOS).200 There is evidence

that NO deriving from nNOS is involved in nociception and pain processing.139,226 Neurogenic

vasodilatation, a possible pathophysiological mechanism involved in migraine442 is mostlikely mediated by nNOS. Furthermore, the selective nNOS inhibitor 7- NitroIndazole (7-NI)

is able to relieve chronic allodynia in spinally injured rats141 and has been shown to reduce

formalin-induced hindpaw licking and acetic acid-induced abdominal constrictions inmice.301,302

Aim of this report was to study the role of nNOS derived NO in an animal model of

trigeminovascular activation.192 Intracisternal infusion of the irritant capsaicin was used tostimulate sensory nerves of the trigeminovascular system. The nNOS inhibitor 7-NI was

used to prevent the production of neuronal derived NO. As nNOS is present in various areas

throughout the brain,16,89,181,248,301 7-NI may affect trigeminovascular nociception and painperception at several levels of pain processing circuitry in the brain. Therefore, no

anaesthetics were used and behaviour of the rats was analysed. Selective activity of the

nociceptive part of the trigeminal system was assessed by measuring the number of cells inthe trigeminal nucleus caudalis, layer I and II (TNC I,II) that were positive for the proto-

oncogene c-fos protein (Fos).


109

Methods

Experiments were approved by the local committee on Bio-Animal ethics of the

University Groningen (FDC 2198) and were performed according to the ethical guidelines for

investigations of experimental pain in conscious animals.503

AnimalsMale Wistar rats (280-350 gr.) were used. Rats were housed individually on a 12

hour light/dark cycle. Food and water were provided ad lib. Surgery was performed 5 or 6

days after arrival of the animals.

SurgeryAll rats received a cisterna magna (CM) cannula 3 days prior to the experiments.

Surgery was performed under semi-sterile conditions. The cannula was made from astainless steel needle (0.6 x 25 mm. 23 G x 1’’; Braun, Melsungen, Germany) of which 6.5

mm was inserted into the brain. Rats were anaesthetized with sodium pentobarbital (60

mg/kg, i.p.) and were placed in a stereotaxic apparatus with incisor bar at –7 mm from thehorizontal plane. Two small holes were drilled into the caudal corners of the interparietal

skull and 2 screws were driven 1.5 mm into the skull. A hole of 1.2 mm was drilled at the

midline of the external occipital crest through which the CM cannula was carefully placed,along the cerebellum, into the CM. Correct placement was confirmed by withdrawal of

cerebrospinal fluid. The cannula was fixed to the skull with dental cement (Kemdemt, Purton

Swindon, UK) and closed by insertion of a metal wire (of the same length as the cannula)within a polyethylene cap to seal the cannula off.

DrugsCapsaicin was kept in stock solution (3.05 gr. in 1 ml vehicle-stock (saline-ethanol-Tween80

8:1:1)) and dissolved 1:40 in saline to yield the 250 nM concentration. Vehicle-stock was

also dissolved 1:40 to serve as control solution for the 250 nM capsaicin solution. Evans blue(0.2%) was added to be able to determine the distribution of the infused solutions after the

experiments. 7-Nitroindazole was kindly provided by Glaxo-Wellcome (London, United

Kingdom) and sonicated in peanut oil in a concentration of 50 mg/kg.

Experimental proceduresRats were injected i.p. with 7-NI or peanut oil 30 minutes prior to the intracisternal

capsaicin or vehicle infusion, a time period resulting in maximal inhibition of neuronal NOS

at the time of capsaicin or vehicle treatment.248 For the infusion, rats were placed in anobservation cage (30 x 30 x 30 cm). Capsaicin or vehicle solution (100 µl) was infused

through the CM cannula over a period of 2 minutes. After infusion, rats were returned to

Chapter 4.2

110

their home cage. The behaviour of rats was recorded on videotape from 5 minutes beforeuntil 10 minutes after the CM infusion.

Perfusion and immunocytochemistryRats were perfused 2 h. after the infusion of capsaicin or vehicle. Immediately

before the transcardial perfusion rats were deeply anaesthetized with sodium pentobarbital

and perfused with 0.9% saline for 1 min, followed by 4% paraformaldehyde (PF) in 0.1 Mphosphate-buffered saline (pH 7.4) for 20 min. After removal of the occipital bone,

placement of the cannula in the CM was confirmed and distribution of the Evans Blue

staining was determined. After the removal, the brains were post-fixed in 4% PF during 24h. Prior to sectioning the brain was cryoprotected by overnight storage in 30% sucrose in0.1 M phosphate buffer (pH 7.4). Forty µm thick coronal serial sections were prepared on a

cryostat microtome at -15°C, and collected in 0.2 M potassiumphosphate-buffered saline(KPBS, pH 7.4) with sodiumazide (0.1%).

Free floating sections were immunocytochemically stained for c-fos protein

according to a standard protocol previously described.192,193 In brief, after preincubation innormal sera and pre-treatment with 0.3 % H2O2, a primary antibody, rabbit anti-Fos

(1:10000; AB5, Oncogene Science inc, Cambridge, UK) was applied overnight at roomtemperature. After thorough washing, the secondary antibody (1:200 biotinylated goat-α-

rabbit IgG (Pierce, Rockford)) was applied for 2 hours. Subsequently, sections were

incubated with the avidine-biotine-peroxidase complex (Vector Labs, Burlingame) for 2 h. at

room temperature. The Ni-enhanced 3,3’-diaminobenzidine tetrahydrochloride reaction wasused to visualize the presence of peroxidase. Intermittent washing was performed with

KPBS, antibodies were dissolved in KPBS with 0.5% triton X-100. All staining procedures

were performed with gentle agitation. Sections were mounted, dehydrated and coverslippedwith DEPEX.

QuantificationC-fos protein

Fos-ir cells in layer I and II of the TNC (TNC I,II) were counted at -1, -2, -3, -4, -5

and -6 mm caudal from obex by an observer blinded from experimental procedures.Sections from -0.5 to -1.5 mm were averaged to obtain the count for the -1 mm level and

so on. The mean of the total TNC I,II was calculated by averaging the Fos expression at the

6 levels.

BehaviourThe behaviour shown in the 5 minutes before, the 2 minutes during the CM infusion

and the 10 minutes after the stimulation was analysed with dedicated software (The

Observer 3.0, Noldus Information Technology, Wageningen, the Netherlands). The first 2


111

minutes immediately after the intracisternal capsaicin or vehicle infusion were not analysed

because we had to uncouple the animals from the microinjector device. Behaviours scoredwere exploring behaviour (exploring and rearing), head grooming / scratching and

immobilization (all forms other than resting). Other behaviours shown include body

grooming, eating and drinking, and resting.

Satistical analysisThe following groups were assembled: Peanut oil + vehicle (Control, n=5), 7-NI + vehicle(7-NI, n=4), peanut oil + capsaicin (Caps, n=4) and 7-NI + capsaicin (7-NI+Caps, n=6).

Effects of 7-NI independent of capsaicin were tested by comparing the 7-NI group to the

Control group. Effects of 7-NI on capsaicin-induced numbers of Fos positive cells in the TNCI,II and behaviour were tested by comparing the 7-NI+Caps group to the Caps group. The

unpaired Student’s t-test was used to test significant differences if data showed normal

distribution, otherwise the Mann-Whitney Rank Sum test was employed. P < 0.05 wasconsidered significant. Fos data are expressed as mean number of positive cells ± S.E.M.

and behavioural data as mean time in seconds spent on each behaviour ± S.E.M.

Chapter 4.2

112

ResultsInclusion criteria

Twenty rats were included in this study in which the EB staining pattern of the dura

mater after intracisternal infusion of capsaicin or vehicle was identical. These rats exhibitedEB staining patterns of the dura of the ventral hindbrain, the caudal brainstem and the

upper levels of the cervical spinal cord.

BehaviourPrior to noxioustrigeminovascular stimulation(fig. 4.2.1)Whereas the decrease of

exploring behaviour by i.p.7-NI was not significant, the

7-NI treated animals showed

significantly moreimmobilization than peanut

oil treated control animals

(94 ± 21 vs. 24 ± 11respectively). Head

grooming/scratching

behaviour (8 ± 5 vs. 41 ± 10respectively) was

significantly reduced after 7-NItreatment. There were no

differences in grooming and

scratching of the bodybetween animals in the 7-NI

and control groups (27 ± 20

vs. 16 ± 7 respectively).

During noxioustrigeminovascular stimulation(fig. 4.2.2)

During the 2 minutes of

intracisternal infusion ofvehicle, the group pre-treated

with i.p. 7-NI showed less

exploring behaviour compared


25

50

75

100

125

150

175

200

225

250 Peanut oil 7-NI

*

*

*

Tim

e (s

ec)

Figure 4.2.1. Effects of the nNOS inhibitor 7-NI on the timespend on different kind of behaviours (mean ± SEM)observed during the 5 minutes prior to intracisternalcapsaicin or vehicle infusion. Peanut oil: n=9, 7-NI: n=10. *is significantly different from peanut oil treated animals (p< 0.05).


10

20

30

40

50

60

70

80

90

100

110

120 Control

7-NI Caps 7-NI+caps

NS NSNS

* *

*

*

*

Tim

e (

sec)

Figure 4.2.2 Effects of 7-NI on the time spend on differentkind of behaviours (mean ± SEM) during the 2 minutes ofintracisternal capsaicin or vehicle infusion. Various groupswere treated with peanut oil + vehicle (Control, n=5), 7-NI+ vehicle (7-NI, n=4), peanut oil + capsaicin (Caps, n=4)and 7-NI + capsaicin (7-NI+Caps, n=6). N.S. means valueis 0 and therefore not shown. * is significantly differentfrom Control (p < 0.05).


113

to the Control group (94 ± 9 vs. 115 ± 2 respectively) and immobilized more (23 ± 9 vs. 0

± 0). Capsaicin decreased the exploring behaviour (Caps: 31 ± 5) and increased bothimmobilization (Caps: 31 ± 7) and grooming and scratching of the head (Control: 0 ± 0,

Caps: 51 ± 6). Capsaicin-induced behavioural changes were not modulated by 7-NI pre-

treatment.

After noxious trigeminovascularstimulation (fig. 4.2.3)

In the 8 minutes after

intracisternal infusion of vehicle, 7-

NI significantly decreased theexploring behaviour (Control: 293

± 25, 7-NI: 174± 35) and

immobilization behaviour showed aconsiderable increase although it

did not reach statistical

significance (Control: 70 ± 42, 7-NI: 244 ± 84). Capsaicin

administration into the CM also

significantly decreased exploringbehaviour (Caps: 35 ± 24) and

induced a significant increase of

immobilization behaviour (Caps:335 ± 42). Head grooming and

head scratching are not altered

by either administration of either7-NI or intracisternal capsaicin

and 7-NI pre-treatment does not

alter the capsaicin-inducedchanges in exploring or

immobilization behaviour.

Fos expression in the TNC I,II(fig. 4.2.4)

Fos expression in the TNCI,II was equally distributed

throughout both the rostro-

caudal and dorso-ventral extendof the TNC. Intraperitoneal 7-NI


50

100

150

200

250

300

350

400

450 Control 7-NI Caps 7-NI+Caps

*

*

*

Tim

e (

sec)

Figure 4.2.3. Effects of 7-NI on the time spend ondifferent kind of behaviours (mean ± SEM) observedduring 8 minutes after infusion of capsaicin or vehicle.Groups were treated with peanut oil + vehicle (Control,n=5), 7-NI + vehicle (7-NI, n=4), peanut oil + capsaicin(Caps, n=4) and 7-NI + capsaicin (7-NI+Caps, n=6). * issignificantly different from Control (p < 0.05).

0

100

200

300

400

500

600

700

800

900 Control 7-NI Caps 7-NI+Caps

*

Fos-

ir (

cells

/sect

ion)

Figure 4.2.4. Number of Fos positive cells (mean ± SEM) inlayer I and II of the trigeminal nucleus caudalis in animalstreated with: Peanut oil + vehicle (Control, n=5), 7-NI +vehicle (7-NI, n=4), peanut oil + capsaicin (Caps, n=4) and7-NI + capsaicin (7-NI+Caps, n=6). * is significantlydifferent from Control (p < 0.05).

Chapter 4.2

114

administration does not affect Fos expression in the TNC I,II (Control: 85 ± 3, 7-NI: 90 ±16). Capsaicin on the other hand, induced a marked increase of Fos expression in the TNC

I,II (Caps: 581 ± 80) but pre-treatment of 7-NI could not significantly alter this Fos

expression (7-NI+Caps: 756 ± 93).


115

Discussion

Intraperitoneal administration of 7-NI significantly decreased exploring behaviour

and grooming and scratching of the head, and it significantly increased immobilization.

Intracisternally applied capsaicin also caused a decrease of exploring behaviour, and anincrease of immobilization, head grooming and head scratching. 7-NI pre-treatment did not

alter the behaviours that were increased or decreased by intracisternal capsaicin

administration. The capsaicin-induced Fos expression in the outer layers of the TNC was notsignificantly altered by the administration of 7-NI, although it was slightly higher.

The reduction of activity observed 30 minutes after i.p. 7-NI treatment is in

agreement with previously reported data showing 7-NI-induced suppression of locomotoractivity.92,93,467 7-NI is able to induce general motor deficits93,141 and sedation181,467 at a dose

of 50 mg/kg, the concentration used in the present experiments. As the behaviour of the

rats did not show any abnormalities that suggested motor deficits, the reduction in headgrooming / scratching behaviour and the induction of immobilization behaviour by 7-NI are

most likely caused by sedation.

Tassorelli and colleagues have shown that TNC Fos expression, induced bysystemic administration of the NO donor nitroglycerin, can be reduced by i.p. pre-treatment

with 7-NI.435 Our experiments show that capsaicin-induced Fos expression in the TNC I,II is

not reduced by i.p. 7-NI pre-treatment. This indicates that nNOS is essential for nitroglycerinto exert its nociceptive action on the trigeminal system but that nNOS is not a necessary

part for trigeminal nociception per se. Trigeminal afferents are capable of ortho and

antidromic conduction of nociceptive signals. Presumably, the induction of Fos in the TNCI,II in the experiments of Tassorelli and co-workers is mediated by antidromically-induced

NO release. In our model of direct stimulation of trigeminovascular afferents, the

orthodromic conduction is responsible for Fos expression in the TNC I,II. This would implythat, whereas neuronal derived NO may be important for the antidromic processing of

trigeminovascular nociception, it is not relevant in the orthodromic processing of pain from

trigeminal afferent to the second order neurons in the TNC I,II.The anti-nociceptive effects of 7-NI demonstrated in other animal models is also

not on direct pain processing but rather at the level of inhibition of sensitisation following

nociception. For example, in mice, 7-NI has antinociceptive effects in the late phase (15-30min) but not in the early phase (0-15 min) after formalin injection into the hindpaw,301,302

suggesting that neuronal derived NO is involved in ‘wind up’ of dorsal horn neurons. This

has been reported before285 and is confirmed by other reports.413 Also, 7-NI is able torelieve chronic allodynia in spinally injured rats,141 further emphasizing a role of neuronal

derived NO in sensitisation processes.

Capsaicin generates hyperalgesia and allodynia in humans54,72,397 and rats.120,403,404

It has been suggested that NO production at the spinal level mediates this effect.487

Chapter 4.2

116

Chemical activation of the meninges is a cause of extracranial mechanical allodynia of theskin.44 The occurrence of allodynia or hyperalgesia after intracranial capsaicin treatment was

not tested in our model, but mechanical allodynia of the skin, caused by meningeal irritation

with capsaicin, may explain why capsaicin treated animals stop grooming and scratching ofthe head shortly after the administration. This implies that nNOS is most likely not involved

in the development of this capsaicin-induced allodynia because 7-NI pre-treatment did not

modify head grooming and head scratching behaviour.There are numerous cerebral nuclei that show increased Fos expression after

intracisternal capsaicin treatment in conscious rats (unpublished results), which also show

positive NOS histochemistry (as measured by NADPH diaphorase histochemistry463). Theseinclude the substantia gelatinosa of the TNC, the nucleus of the solitary tract, the lateral

parabrachial nucleus, the dorsal raphe nucleus, the basolateral amygdala, the supraoptic

nucleus of the hypothalamus, the paraventricular nucleus of the hypothalamus and thecentral medial thalamic nucleus.463 Despite these possible cerebral targets for 7-NI and the

observed sedation caused by 7-NI administration, we observed no effects of 7-NI pre-

treatment on acute trigeminovascular nociceptive response behaviour. This suggests thatnNOS inhibition also is not essential in the sensory processing of trigeminal pain

downstream from the TNC and that the sedative effects of 7-NI are easily ‘overruled’ by

noxious stimulation with capsaicin.In conclusion, our results do not provide evidence for a role of neuronal derived

nitric oxide in the orthodromic processing of acute trigeminovascular pain.

Section 5

General discussion

Section 5

118

Summary of the resultsIn chapter 2.1 we showed that trigeminovascular activation by intracisternal

infusion of capsaicin in the conscious rat elicits immobilization behaviour, head grooming,

head scratching, and escape behaviour. The concentration of the capsaicin solutiondetermined the behavioural response type.

In chapter 2.2 the cerebral activity patterns, as revealed by Fos

immunocytochemistry, following trigeminovascular activation were determined. Especiallyareas that are known to process or inhibit pain and areas that are involved in autonomic

control were activated. These include the dorsal raphe nucleus and the locus coeruleus, two

areas that have been speculated to be pattern generators in migraine.In chapter 3.1 the relationship between the immunesystem and migraine was

investigated by reviewing the literature that supplies measurements of various

immunological factors in migraineurs. Although different immunological parameters havebeen reported altered, there is no apparent pattern of distinct immunological pathology

present. The only consistent finding appears to be elevated plasma histamine levels in

migraineurs independent of the attack and may be related to increased spontaneoushistamine release by leukocytes. Increased plasma histamine levels are not related to a

hypersensitive immunesystem but are more likely to be due to an increased susceptibility or

sensitivity for infectious diseases.In chapter 3.2 we studied the effect of a bacterial infection on trigeminovascular

nociceptive processing in the conscious rat. Low concentrations of injected

lipopolysaccharides (LPS) enhanced the capsaicin-induced immobilization behaviour andhigher concentrations of LPS increased the capsaicin-induced Fos expression in the outer

layers of the TNC. Thus infections may cause a headache of the highest intensity in

migraineurs by inducing hyperalgesia in the trigeminovascular system.In chapter 4.1 and 4.2 we examined whether the somatostatin analogue octreotide

and the neuronal nitric oxide synthase inhibitor 7-NitroIndazole modulated the

trigeminovascular system through central nervous system mediated mechanisms. Whereasboth compounds affected behaviour independently of trigeminovascular stimulation, they

were not capable of altering acute trigeminovascular nociceptive processing (although a

very small inhibitory effect of octreotide was found in the caudal most part of the TNC).

Conscious vs. anaesthetized?After four years experience with a model of trigeminovascular stimulation in the

conscious rat, the question arises, which benefits were provided by the use of the

unanaesthetized animal model. In chapter 2.1 and 2.2 we were able to analyse behaviour

and cerebral activity patterns associated with trigeminovascular nociception, which would beimpossible in anaesthetized animals. The experiments revealed that activation of the locus

coeruleus and dorsal raphe found in migraineurs during and shortly after the attack may

Discussion

119

well be related to the pain of the attack instead of being a pattern generator. In chapter

3.2, the unanaesthetized conditions enabled us to show that low concentrations of LPS,which had physiological but no behavioural effects, increased the immobilization behaviour

caused by trigeminovascular stimulation. Only the use of conscious animals enabled us to

obtain evidence for central nervous mediated actions of compounds that do not modulatecentral pain processing after intracisternal capsaicin injections (chapters 4.1 and 4.2)

All experiments presented thus benefited from the absence of anaesthetics in one

way or the other. It is clear that for studying pain processing downstream from the TNC,anaesthetics limit interpretation to a large extent. Most animal models of trigeminovascular

stimulation have used anaesthetics and studied the processing of nociception between the

extracerebral vessels in the meninges and the TNC. Valuable information regarding theorigin, processing and inhibition of trigeminovascular nociception was gained from these

models, and most likely, the use of anaesthetics did not lead to misinterpretation of these

experiments. This implies that the use of conscious animals is only indicated for studiesaddressing topics such as the processing and modulation of trigeminovascular nociception

downstream from the TNC. Only in such studies, benefits may overrule the ethical aspects

associated with inflicting painful stimuli to conscious animals. The number of animals, theintensity of the stimulus and the duration of the stimulus should, however, always be kept

as low as possible.

Behaviour in-depthCombining the data from section 2 and 3 of the controls and capsaicin treated

animals (10, 100, 250 and 1000 nM) it can be shown that the contribution of the variousbehaviour types as a measure of trigeminovascular stimulation depends on the

concentration of the capsaicin solution used. The lower dosages of 0 to 200 nM

predominantly decreased exploring behaviour, and increased immobilization behaviour.Dosages between 200 and 400 nM decreased exploring behaviour but in these cases it is

associated with an increased head grooming, head scratching and escape behaviour.

Dosages above 400 nM do not change the behavioural response. These findings are basedon the assumption that exponential decay and a sigmoidal (dose response) curve is the best

way to describe the dose response effect of exploring behaviour and the active type of

capsaicin-induced behaviours, respectively. The average percentage of the total time of 2minutes of infusion that can be explained by these two types of behaviours combined with

immobilization is 94%. The immobilization behaviour curve is extrapolated from the other

two curves.If a sigmoidal (dose response) curve is used to model the average number of Fos

positive cells in the TNC I,II, induced by increasing concentrations of capsaicin (with the

assumption that the number of TNC Fos-ir cells is maximal at 1000 nM capsaicin), the doseof capsaicin that induces 50% of the maximal Fos expression (EC50) is 465 nM. The EC50 of

Section 5

120

the sigmoidal curve for the active behaviours generated by intracisternal capsaicin

administration is 284 nM, and it is even smaller for exploring behaviour. Depending on thedose of capsaicin, exploring behaviour, head grooming/scratching & escape behaviour, and

Fos expression would be the best parameters to study the effects of increasing dosages of

noxious compounds on the trigeminovascular system.One final comment has to be made. Remarks about relationships between

behavioural responses and capsaicin dosages are valid only for the experiments described in

section 2 and 3 in which we used animals from the same supplier. Studies conducted laterusing animals from a different supplier were more sensitive to capsaicin. This difference of

pain sensitivity within species has been examined extensively for mice recently, showing

that mice obtained from different breeding programs show up to 50 times difference in painthresholds.298 Dose response curves for behaviour and cerebral Fos patterns after noxious

stimulation, therefore, should be established every time there is a change of supplier or

strain of rats.

Peripheral vs. central?The results obtained from the experiments in section 4 suggest that the nNOS

inhibitor 7-NI and the somatostatin analogue octreotide do not modify orthodromic

Figure 5.1 Model predicting dose-response relations between the intracisternally administeredamount of capsaicin and the time spent on each type of behaviour during the infusion.Individual points are obtained from experiments described in section 2 and 3.

0 200 400 600 800 1000

0

20

40

60

80

100

120 Exploring behaviour

Fit: exponential decay

Head grooming / scratching & escape behaviour

Fit: sigmoidal (dose response)

Immobilization behaviour

Fit: exptrapolated from both other fits

Tim

e (s

ec)

Dose of capsaicin

Discussion

121

nociceptive processing of trigeminovascular nociception whereas these compounds exhibited

central activity. Several compounds have inhibited orthodromic trigeminal nociceptioneffectively at the level of the TNC I,II in animal models. These include compounds that act

agonistically on serotonergic receptors, especially 5HT1B, 5HT1D and 5HT1F

receptors,73,79,129,157-159,185,212,297,419,419 block the NMDA receptor,61,296 inhibitcyclooxygenase,186 act through GABAA receptors75,77 and block the neurokinin-1

receptor.61,78 (blockade of the latter receptor has also been reported to be unsuccessful to

inhibit trigeminal nociception131). Many of these drugs have also been shown effective inmigraine treatment. The ergot alkaloids,112,237 naratriptan,35,272 sumatriptan,98,273,362,465

rizatriptan,466 zolmitriptan,351,406,498 NSAIDs1,237 and valproate (see395) have all been used

successfully to treat migraine headache. The question arises whether their action is indeedat central sites or whether it is at peripheral sites, for example at vascular receptors or at

the perivascular trigeminal afferent terminal sites. Despite a poor penetration of the blood

brain barrier (BBB), the efficacy of sumatriptan98,102,273,315,362,465 in aborting the headache, isin the same range of efficacy as the supposedly centrally acting triptans like

naratriptan,35,272 rizatriptan466 and zolmitriptan351,406,498 (although recurrence, onset of relief

and side effects may differ). This strongly implicates that a contribution of centralserotonergic receptors is not necessary for mediation of headache relief.

It has been suggested that BBB leakage occurs during the headache phase of

migraine, which would explain the comparable efficacy of the various triptans which havedifferent BBB penetration. Also, BBB leakage could explain why sumatriptan is only effective

when administered during the headache phase of a migraine attack. Gadolinium - Magnetic

Resonance Imaging (MRI) scans of the trigeminal nucleus of 6 migraineurs fulfilling IHScriteria, however, showed no signs of BBB-leakage during the migraine attack (unpublished

results). Two of these migraineurs were treated with subcutaneous sumatriptan immediately

following the MRI-scan and they experienced headache relief within 15 minutes after thistreatment. We therefore believe that the action of sumatriptan in relieving migraine

headache is peripheral and not at 5HT1b/d receptors located for example in the TNC. The

additional beneficial effect of central penetration by the ‘next generation’ triptans most likelyis relatively small.

The potential side effects of centrally acting triptans may even counterbalance the

beneficial effects. Apart from the outer layers of the TNC and maybe a border layer in theventrobasal thalamic nuclei, trigeminovascular stimulation induces activation in many areas

that are not specific for processing trigeminovascular nociception (see chapter 2.2).

Inhibition of nociceptive processing between perivascular trigeminal afferent terminals andthe cerebral cortex will be most effective and selective when it is able to prevent activation

of the perivascular afferent. If a good efficacy can be achieved by intervention in peripheral

mechanisms, for example by vasoconstriction or inhibition of PPE, additional central effectsmay be undesirable. The endogenous supraspinal pain inhibition pathways to the TNC I,II

Section 5

122

are most likely already maximally activated during a severe migraine attack. Therefore,neuropeptide modulation of pain transmission mediated by the classical neurotransmitters

may not contribute to reduce signal transduction. The efficacy of co-transmitter modulation

in anaesthetized animal models most likely could be shown because the anaestheticsprevented the activation of the endogenous pain modulating systems.

Considering the high efficacy of migraine treatment with drugs that act acutely but

do not penetrate the BBB, and the relatively low additional advantage of centrally actingcompounds (combined with high risk of side effects of centrally active drugs), little room

remains for improving acute migraine treatments.

Preventing the migraine attack would be the most profitable approach for patients.Besides behavioural therapy aimed at attack prevention (ea. avoiding certain foods,

flickering lights, exercise etc.) there would be room for pharmacological mediated

prevention. Current pharmacological prevention include β-adrenergic blockers,antidepressants, calcium channel antagonists, serotonin antagonists, anticonvulsants and

NSAIDs (see396). The drugs with the highest efficacy (β-blockers, methysergide, trycyclic

antidepressants, monoamine oxidase inhibitors and valproate) however, also have thehighest amount of side effects (see396). In contrast to the acute treatment of the attack, the

primary target side of action of most prophylactic compounds are receptors located inside

the brain (see124). Prevention of PPE at perivascular trigeminal nerve terminals may also bea target for attack prevention by some drugs.76,124

Migraine prevention can only be optimized when the pathophysiological

mechanisms occurring hours to days before the headache are well understood. This phasemay be characterized by prodromes in some migraineurs and has received little attention in

migraine research. In the review presented in chapter 3.1, all studies reported

immunological parameters obtained during the headache phase of the migraine attack andnot the day preceding the attack. If immunological and physiological alterations contribute

to migraine generation, a good period for observing physiological changes would be 24 hrs.

before the start of the headache. The report that described the beneficial effect ofHelicobacter Pylori eradication on migraine attack frequency, duration and intensity116 shows

that prophylactic treatment in some migraineurs may also involve treatment of non-cerebral

pathology.

References

123

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Samenvatting

147

SamenvattingMigraine is een aandoening waaraan ongeveer 6% van de mannen en 16% van de

vrouwen lijdt. Een migraine aanval wordt over het algemeen gekarakteriseerd door een

unilaterale, kloppende hoofdpijn, welke gepaard gaat met misselijkheid en een afkeer van

licht en geluid. In een minderheid van de patiënten wordt de hoofdpijn-fase voorafgegaandoor een aura: een verstoring van de sensorische waarneming (meestal visueel), die 20 tot

30 minuten voor het begin van de hoofdpijn start en vlak ervoor stopt. Een deel van de

migraine patiënten voelt de aanval al een dag van te voren aankomen doordat ze lastkrijgen van geirriteerdheid, gapen, vermoeidheid en veranderd eet en drink gedrag; dit zijn

de zogenaamde prodromen.Het onderzoek naar de pathofysiologische processen die ten grondslag liggen aan

migraine vindt zowel op klinisch als pre-klinisch niveau plaats en neemt, beoordeeld naar

het aantal wetenschappelijke artikelen erover, sinds de jaren 60 gestaag toe. Desondanks is

het mechanisme dat verantwoordelijk is voor het ontstaan van migraine nog onbekend. Welzijn er verschillende theorieën over dit mechanisme ontwikkeld. De meeste theorieën gaan

er van uit dat als laatste stap het trigeminovasculaire systeem wordt geactiveerd. Dit

systeem is het complex van pijnzenuwen van trigeminale origine, welke intracraniaalgeassocieerd zijn met de grotere vaten van het brein en de dura mater; één van de

hersenvliezen. De theorieën verschillen met name in de manier waarop dit

trigeminovasculaire systeem wordt geactiveerd. Oorzaken van migraine worden gezocht invaatverwijding, neurogene ontstekingsprocessen, en/of afgegifte van stikstofmono-oxide of

vaso-actieve amines. Recentelijk heeft men ook aangetoond dat genetische afwijkingen in

een calciumkanaal tot een bepaalde vorm van migraine leiden.Omdat de hoofdpijnfase het meest invaliderende aspect van de migraine aanval is

wordt in het pre-klinisch onderzoek met name deze fase onderzocht. Stimulatie van

intracraniaal gelegen trigeminale pijnzenuwen op chemische, electrische of mechanischewijze worden gebruikt om de hoofdpijn na te bootsen in proefdieren. Als parameter voor

activiteit van het trigeminale systeem wordt onder andere de neuronale activiteit in de

trigeminaal nucleus caudalis (TNC) gebruikt. De buitenste lagen van de TNC zijn hetprimaire station in de hersenen waar trigeminale pijnzenuwen eindigen. Middels electrische

afleiding of immunocytochemische bepaling van eiwitten die tot expressie komen als gevolg

van verhoogde activiteit van neuronen (zoals Fos, het eiwit van het proto-oncogen c-fos),kan de activiteit in deze nucleus worden gekwantificeerd.

Tot nu toe werden hoofdpijnmodellen altijd ontwikkeld in genarcotiseerde dieren.

De perifere nociceptieve processen in de hersenvliezen die aanleiding zijn voor hetpijnsignaal zijn waarschijnlijk niet gevoelig voor anesthesie. De centrale verwerking van

Samenvatting

148

pijnsignalen wordt echter wel beïnvloed door de anesthesie. Een diermodel voor migrainehoofdpijn waarbij geen anesthesie wordt toegepast, geeft ons de mogelijkheid om de

centrale verwerking van hoofdpijnsignalen te bestuderen en is met name interessant

vanwege de ontwikkeling van anti-migraine farmaca met een (additionele) centrale werking.Het ethische aspect van pijninductie bij niet genarcotiseerde proefdieren wordt door ons

gezien als een belangrijk, maar moeilijk te vermijden, nadeel.

Doel van het onderzoek beschreven in dit proefschrift is de ontwikkeling van eenproefdiermodel voor hoofdpijn in de ongeanestheseerde rat om vervolgens de centrale

verwerking van trigeminovasculaire nociceptie te bestuderen en de fysiologische en

farmacologische modulatie daarvan.Sectie 1 van dit proefschrift geeft een algemene inleiding over migraine, waarin

enkele theorieën over de pathofysiologie van migraine de revue passeren en dieper wordt in

gegaan op de verschillende proefdiermodellen die worden gebruikt voor bestudering vanmechanismen die betrokken zijn bij het ontstaan van trigeminovasculaire pijn.

Sectie 2 van dit proefschrift beschrijft het gedrag en de cerebrale activiteit die

geassocieerd zijn met trigeminovasculaire pijn in de rat. Deze sectie omvat 2 hoofdstukken.Hoofdstuk 2.1 beschrijft het model dat is gebruikt om migraine hoofdpijn in

ongeanestheseerde ratten na te bootsen. Intracraniale trigeminale pijnzenuwen werden

gestimuleerd door verschillende concentraties capsaicine (de scherpe, actieve stof in rodepepers, die C vezels stimuleert) te infuseren in de cisterna magna, een ruimte gelegen

achter het vierde ventrikel en het cerebellum die een ruime hoeveelheid cerebrospinale

vloeistof bevat. Dit werd gedaan middels een permanente cisterna magna cannule, die 3dagen voor het experiment werd geplaatst. Voor het 2 minuten durende infuus van 100 µl

oplosvloeistof of 10, 100 of 1000 nM capsaicine werden de ratten in een nieuwe kooi

geplaatst en het gedrag werd op video opgenomen voor gedragsanalyse. Na 2 uur werdende ratten getermineerd waarna de hersenen werden onderzocht op de expressie van Fos.

Het aantal Fos positieve cellen in de buitenste lagen van de TNC werd gekwantificeerd.

Exploratie van de kooi was een karakteristiek onderdeel van het gedrag van dierendie oplosmiddel kregen toegediend. Dit exploreer gedrag werd dosis-afhankelijk verlaagd bij

infusie van toenemende concentraties capsaicine. Hiervoor in de plaats gingen ratten

immobiliseren en de kop wassen en krabben. Het experiment laat zien dat stimulatie vanintracraniale pijnzenuwen leidt tot extracraniale "referred pain" sensatie die was- en

krabgedrag induceert. Extracraniale overgevoeligheid van de huid van het hoofd wordt ook

bij migraine patienten waargenomen. De hoeveelheid Fos positieve cellen in de buitenstelagen van de TNC was alleen significant verhoogd na toediening van de hoogste

concentratie capsaicine. Dit laat zien dat de gedragseffecten van capsaicine toediening al

waarneembaar zijn als verandering van de neuronale activiteit in de TNC niet meetbaar ismet behulp van Fos expressie. Dit valt mogelijk te verklaren doordat de temporele resolutie

van Fos niet gelijk is aan die van de parameter gedrag.

Samenvatting

149

Hoofdstuk 2.2 beschrijft het cerebrale Fos expressie patroon gevonden na

intracisternale infusie van 250 of 1000 nM capsaicine. Gebieden die verhoogde Fosimmunoreactiviteit lieten zien na 250 en/of 1000 nM capsaicine waren naast de buitenste

lagen van de TNC verschillende delen van het "limbische systeem" (nucleus tractus

solitarius, area postrema, parabrachiale nucleus, amygdala, periaqueductale grijs,hypothalamus, intralaminaire thalamische nuclei, insulaire cortex) en gebieden betrokken bij

supraspinale pijninhibitie (locus coeruleus, raphe dorsalis, raphe magnus) en

pijngewaarwording (delen van de primaire somatosensorische cortex). Van bijna al dezegebieden is bekend dat ze een rol bij pijnmodulatie (kunnen) spelen, maar het valt niet uit

te sluiten dat gebieden geactiveerd werden door de fysiologische en gedragsmatige

responsen na trigeminovasculaire nociceptie. Dat de locus coeruleus en raphe dorsalis ookduidelijk geactiveerd worden door de trigeminovasculaire pijnprikkel pleit tegen een

belangrijke rol van deze nuclei bij het ontstaan van een migraine aanval, zoals wordt

gesuggereerd door neuro-imaging studies die zijn uitgevoerd bij migraine patiënten tijdensde aanval.

Sectie 3 bevat 2 hoofdstukken waarin de relatie tussen immunologische factoren

en migraine een belangrijke rol speelt. Hoofdstuk 3.1 is een overzichtsartikel overimmuunsysteem functie in migraine. Er zijn meerdere (indirecte) aanwijzingen dat het

immuunsysteem betrokken is bij de migraine pathofysiologie. Atopsiche ziekten zoals

exceem en asthma, komen vaker voor bij migraine patiënten, evenals een verhoogdegevoeligheid voor infecties. Bovendien is aangetoond dat de frequentie, duur en intensiteit

van de migraine-aanvallen afnemen wanneer de patiënten behandelt worden voor de

bacteriele Helicobacter Pylori infectie. In het overzichtsartikel worden studies besproken diehet functioneren van het immuunsysteem in migraine patiënten hebben onderzocht. Hierbij

komen immunoglubulines, complement factoren, histamine, cytokines, en immuun systeem

cellen aan de orde.Uit het review blijkt dat er geen eenduidige, goed gedefinieerde immunologische

afwijking aanwezig is in migraine patiënten, maar dat sommige immunologische parameters

inderdaad veranderd zijn. Verhoogde plasma histamine niveau’s, verhoogde spontaneafgifte van histamine door leucocyten, veranderde (mogelijk onderdrukte) functie van

lymphocyten en verlaagde fagocytotische capaciteit van monocyten en polymorphnucleaire

cellen worden gevonden bij migraine patiënten. Deze veranderingen duiden eerder op eenonderliggende persistent aanwezige infectie dan op een zich herhalende atopische

overgevoeligheidsreactie van het immuunsysteem.

In hoofdstuk 3.2 wordt het in hoofdstuk 2.1 beschreven diermodel gebruikt omte onderzoeken hoe infecties trigeminovasculaire pijn beïnvloeden. Infecties werden

nagebootst door een injectie met lipopolysacchariden (LPS – componenten van de celwand

van gram-negatieve bacterïen) 5 uur voor de trigeminovasculaire stimulatie met capsaicine.Gedrag van het dier en Fos expressie in de TNC werden gekwantificeerd als parameters

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voor trigeminovasculaire nociceptie. Een lage concentratie van LPS potentieerde hetcapsaicine geinduceerde immobilisatie gedrag zonder dat het de capsaicine geinduceerde

Fos expressie in de buitenste laag van de TNC beïnvloedde. Een hoge concentratie LPS

verhoogde wel de capsaicine geïnduceerde neuronale activiteit in de TNC. Deze effecten vanLPS op capsaicine gevoelige intracraniale zenuwen worden waarschijnlijk veroorzaakt door

cytokines (de signaalstoffen van het immuunsysteem), die via verschillende wegen de

gevoeligheid van de sensorische zenuwen kunnen beïnvloeden. Overgevoeligheid vantrigeminale zenuwen verklaart mogelijk waarom migraine patiënten de hoogste

hoofdpijnintensiteit rapporteren tijdens een infectie.

In sectie 4 is geprobeerd door toediening van centraal actieve farmaca detrigeminovasculaire nociceptie te beïnvloeden. Hoofdstuk 4.1 beschrijft experimenten met

een langwerkende somatostatine analoog: het octreotide. Deze stof is al met succes getest

bij enkele migraine patiënten maar is weinig lipofiel en ondervindt dus problemen bij hetpasseren van de bloed-hersen barrière. Het precieze werkingsmechanisme van octreotide is

nog onbekend maar er is een uitgebreid systeem van somatostatine positieve vezels en

receptoren aangetoond in de TNC, en andere gebieden van het brein die een rol spelen bijde centrale verwerking van pijn. Daarom is het waarschijnlijk dat nieuwe, meer lipofiele

somatostatine agonisten, via deze gebieden de trigeminale pijnverwerking zouden kunnen

beïnvloeden. Daarom werden effecten van intracisternaal toegediende octreotide getest inhet model voor trigeminovasculaire nociceptie zoals beschreven in hoofdstuk 2.1.

Octreotide, toegediend 10 minuten voor de infusie van het oplosmiddel, veroorzaakte

(buiten capsaicine om) een verhoging van het wassen en krabben van de kop, wat centraalnerveuze werking van octreotide via deze toedieningswijze ondersteunt. Octreotide kon

echter niet het capsaicine geinduceerde gedrag, noch de toename van de Fos expressie in

de TNC reduceren. Dit duidt er op dat er geen belangrijke primaire rol is voor de centralesomatostatine receptoren bij de verwerking van trigeminovasculaire pijn.

In hoofdstuk 4.2 worden de bevindingen van de neuronale stikstof mono-oxide

synthase (nNOS) remmer 7-Nitro-indazole (7-NI) in het trigeminovasculaire nociceptiemodel beschreven. 7-NI toonde al anti-nociceptieve werking in verschillende pijn modellen.

Aangezien het makkelijk de hersenen binnenkomt, werd het 30 minuten voor de activatie

van het trigeminale systeem intraperitoneaal toegediend. De gedragsstudie toonde eenverhoogde immobilisatie van de dieren na 7-NI toediening en duidelijk minder wassen en

krabben van de kop. Capsaicine-geïnduceerde gedragsveranderingen (immobilisatie, kop

wassen en krabben) werden niet beïnvloed door 7-NI. In overeenstemming daarmee bleekook de capsaicine geïnduceerde Fos expressie in de TNC niet significant verlaagd te worden

door 7-NI. Deze resultaten pleiten tegen een rol voor, uit neuronen afkomstige, stikstof

mono-oxide in trigeminovasculaire pijnverwerking.Sectie 5 is de algemene discussie van dit proefschrift. Hierin wordt het nut van

proefdieronderzoek voor het begrip van (migraine) hoofdpijn bediscussieerd mede in relatie

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tot het "negatieve" ethische aspect. Er wordt dieper ingegaan op het belang van

gedragsstudies als parameter voor de kwantificering van trigeminovasculaire pijn en debetekenis van centraal werkende anti-migraine farmaca voor de acute en/of profylactische

bestrijding van migraine.

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DankwoordDit boekje is het resultaat van zo’n 4 jaar onderzoek bij de Biologische Psychiatrie.

Het was een fantastische tijd op die afdeling. Als je migraine onderzoek doet op een

afdeling psychiatrie hoor je je eigenlijk een vreemde eend in de bijt te voelen. Niets was

minder waar. Vele mensen hebben me geholpen om het onderzoek goed te laten verlopenen misschien wel net zo belangrijk, om het daar super gezellig te maken! Iedereen die

hieronder staat genoemd, heeft op zijn of haar manier bijgedragen aan het boekje en dit is

de beste plek om jullie allemaal te bedanken. Het dankwoord is dus gewoon lekker lang!(wat inhoudt dat dit een zeer goed gelezen proefschrift wordt....)

Als eerste wil ik Jaap bedanken. Als hoofd van het lab en promotor van mijn

onderzoek ben je essentieel geweest tijdens die 4 jaar. Je stond altijd klaar voor me als ik jenodig had. Bedankt daarvoor!

En dan Gert. Gert, je had de directe begeleiding van mijn project in handen en ik

weet maar al te goed dat ik enorm lucky ben geweest met zo’n directe begeleider. Je gafme duidelijk het gevoel dat ik een ‘team’ vormde met je. Je deur stond altijd open om te

discussieren over alles wat met het onderzoek te maken had. Er was absoluut geen

drempel. Je bent ontzettend enthousiasmerend, weet overal de positieve kant eruit tepakken, en als ik een geschreven stuk bij je op bureau legede had ik het een dag later

volledig gecorrigeerd terug. ‘De Dip’ waar vele AIO’s me voor waarschuwden, heb ik niet

gehad, en dat komt mede doordat je, waar je maar kon, het onderzoek fasciliteerde. Ook dejaarlijkse congresbezoeken met je in Amerika waren Big Fun (Universal, Walt Disney en The

Mall... o ja de congressen waren ook leuk....). Gert, bedankt voor die fantastische tijd!.

De klinische kant van de begeleiding heeft Pim op zich genomen. Pim, zonder jouwas het project niet eens van de grond gekomen. Je helicopter view over het project

tezamen met de directe link die je vormde met de kliniek en naar Glaxo-Wellcome toe (waar

je me zonder voorbehoud gebruik van liet maken) vormde een belangrijke motor achter hettot stand komen en het draaiende houden van het project. Daarnaast ben je zonder twijfel

degene die het best weet waar de meest exclusieve eetgelegenheden in Groningen zitten!

Pim, bedankt!Dat was de hulp die ik van bovenaf heb gehad. Praktisch gezien heb ik verreweg

de meeste hulp gehad van Mary. Mary, ik had me geen betere en vooral geen leukere

analiste kunnen wensen. Ik had al snel in de gaten dat je een top analiste was: supersecuur, netjes en iemand die van aanpakken wist en eigen initiatief had. Ik kon je gewoon

je gang laten gaan, je zat zo goed in het onderzoek dat alles supersnel en efficiënt verliep.

Daarnaast ben je integer en heb je een aanstekelijk gevoel voor humor. Mary, ik bewonderje voor de manier waarop je je privé omstandigheden wist te combineren met het werk.

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Ondanks de niet altijd even makkelijke situatie thuis, heb je ontzettend veel werk verzet

voor ons project, en zonder jou had dit boekje hier nu niet gelegen. Ere wie ere toekomt.Mary, bedankt!

De ondersteuning vanuit Glaxo-Wellcome, die naast de ruime financiële sponsoring

vooral bestond uit een prettige samenwerking met Mirjam en (in het begin) Annelies wil ikook bijzonder bedanken. Jullie positief kritische en flexibele benadering van het project vond

ik enthousiasmerend en was een goede basis om het project op te laten bloeien. Daarnaast

heeft de goede samenwerking met jullie m’n interesse gewekt voor de farmaceutischeindustrie en mede daardoor ben ik daar nu aan het werk. Bedankt!

En dan zijn er natuurlijk nog de studenten! Maar liefst 7 studentes (en Ed, grinnik)

hebben me een half jaar of langer geholpen door hun afstudeeronderwerp bij me te doen.Sommigen met succesvolle resultaten, sommigen met minder succesvolle resultaten, maar

altijd was het super stimulerend! In chronologische volgorde I present: Sjoukje, Linda, Ed

(knippen en plakken, Peppi en Kokki), Margriet, Kerstin, Marianne (tuuterdetuut, ben je diebloemenzaak nu al begonnen?), Gea (wat doen die kamelen in m’n kamer?) en Marieke.

Allemaal ontzettend bedankt voor jullie enthousiaste bijdrage aan dit boekje!

En dan alle vaste mensen op het lab, die in meer en mindere mate praktischhebben bijgedragen aan dit onderzoek, maar die er vooral voor zorgden dat het zo gezellig

was op het lab. Folkert, (hoe is het met je Baan...?, niet teveel kranten lezen he!) en Kor

(hou je de ‘history’ nog wel eens bij? hehe), bedankt voor jullie praktische ondersteuning enjullie gezelligheid daar buiten om. Mensen als jullie zijn op een afdeling hard nodig, de

zogezegde smeerolie in de machine van het onderzoek. Bedankt!

Tineke K (denk nou eindelijk eens een keertje ook aan jezelf...), Tineke S (onzemeest enthousiaste deelneemster van de Labdag!), Willy (50 is echt een prachtige leeftijd...,

ik heb trouwens nog een adresje waar ik Extacy kan krijgen..., geinteresseerd?),

weegkamerschoonhouden!-Rikje en Lammy. Hartstikke bedankt voor jullie gezelligheid!Ook alle AIO’s en andere collega’s van Labipsy wil ik bedanken. Ik ga jullie niet

allemaal noemen (vergeet ik ook niet iemand....) maar wil special thanx uit laten gaan naar

Jaqueline (nu er kids komen verwacht ik dat je verstandig wordt en eindelijk inziet dattrouwen toch veel leuker is dan samenwonen!), ‘statistics is his middle name’-Bill, iseekyou-

Marjan en Joke (ik mis de lunch, schepping en evolutietheorie zijn wel degelijk

verenigbaar!).Harm, Helma en Siert, ik reken jullie tot mijn beste vrienden. Dat ik paranimf

mocht zijn bij jullie promoties vond ik een grote eer. Harm en Siert, jullie hebben me

enthousiast gemaakt om ook promotieonderzoek te gaan doen. Het waren nu-al-legendarische tijden! Touchables en ranzige spare-ribs bbq’s staan nog goed in m’n

geheugen gegriefd. Harm, jij was de rustige en bedachtzame twijfelende twijfelaar van de

2, Siert de snelle en de ‘zorg dat je alles goed geregeld hebt’ cola-drinker... Jullie ‘veteranen’tips zijn zeer welkom geweest tijdens de start van m’n onderzoek. Harm, niet meer

154

proberen in te loten voor geneeskunde ok? en Siert, je weet dat ik zo een oppas kan

regelen dus... . Bedankt voor die fantastische begin periode bij Labipsy!Helma, je bent een verhaal apart. Toen Harm en Siert vertrokken waren was jij m’n

enige (en enigste!) overgebleven maatje van de club van 4. Je deur stond altijd open als ik

weer eens in Grûningen moest blijven pitten en vele diepzinnige gesprekken en slapgeouwehoer hebben we tijdens die periode gehad. Je turbulente dynamische en onrustige

leven was en is een mooi tegenwicht voor mijn nogal burgelijke inslag. ICQ is een goed

bindmiddel tussen ons nu je zo ver weg zit (en ook al die CD’s die je voor me uit Hong Kongmeebrengt....hehe). Na de promo zal ik de telefoonkosten weer eens flink gaan opjagen!

Helma, Bedankt!

Dan mijn dierbare paranimfen. Michiel, eerst al ‘best man’ bij m’n trouwerij, nuparanimf. En terecht. Je stond altijd klaar voor me als ik je nodig had. Ontelbare keren kon

ik bij je overnachten toen ik naar Zwolle was verhuisd. Het maakte je niets uit. Hoevaak ik

wel niet kip met gebakken aardappelen met Mayo en bloemkool heb gehad, ik weet hetniet.... De stap avonden, het computeren en de casino weekenden zorgden voor de nodige

ontspanning tijdens die drukke periode. Je beste eigenschap is de ‘hoe geniet ik het meest

in de minste tijd’ houding en bij zo iemand in de buurt is het altijd goed vertoeven. Michielbedankt! (en ik zal je niet meer duwen als je tegen een kerk aan staat te plassen...).

Camillo, ik spreek je veel en veel te weinig en dat is doodzonde want iemand die

altijd zo’n superhumeur heeft (die smile is niet van je gezicht te krijgen....) moet je vakerzien. In Almere zit hoop ik een Thai (en anders kook ik het voor je!), we gaan absoluut mee

op wintersport next winter (nu heb je het zwart op wit!) en dat weekendje survivalen in de

Ardennen moet er nu ook maar eens van komen... . Bedankt dat je m’n paranimf wilt zijn!En dan een hoop vrienden en familie die met de regelmaat van de klok voor het

broodnodige relaxen zorgden en altijd geinteresseerd waren in het verloop van het

onderzoek: de Hazen: Leotter, C’er en de Camel bedankt dat jullie je zo gewillig naar deslachtbank laten lijden met C&C en Quake, blijf oefenen..., trouwe Susan (hoe zit het met

de...? hehe), K’tje (Harm getemd, dacht dat het nooit zou gebeuren!), Anja (met een

nieuwe dame in huis zal het vechten worden voor Sierts aandacht...), Penny en Raoul (hetis echt leuk om goeie buren te hebben!), Karin (promoveren, go for it!) en Gert (hou je haar

wel een beetje rustig...?), Pa en Ma Proper (altijd geinteresseerd en trots), Stefan (m’n

stoere broer en de beste moppentapper die ik ken) en Mieke (nieuwe kleren?), Assie (m’nknappe, zichzelf altijd onderschattende, lieve zus) en Koos (welkom!), allemaal ontzettend

bedankt!

Als voorlaatste mijn lieve Pa en Ma. Ik heb ontzettend genoten van hetpromotieonderzoek en dat kan alleen maar als je lieve ouders achter je hebt staan die je

blindelings vertrouwen (‘als jij denkt dat het goed is, is het goed...’) en die trots op je zijn.

Jullie hebben me altijd gemotiveerd om te leren, juist als ik er geen zin in had of het nut meontging. ‘leer nu maar, jij krijgt tenminste de kans, later ben je blij dat je het gedaan hebt’,

zeiden jullie vaak. Dankzij jullie heb ik die kansen gegrepen (jullie hadden helemaal gelijk!)

155

en het uiteindelijke resultaat van al die jaren leren ligt voor jullie. Het is aan jullie

opgedragen. Bedankt!Het beste moet je voor het laatst bewaren. Eefje, jij hebt heel mijn promotie

onderzoek van begin tot eind van dichtbij meegemaakt en het meest dankbaar ben ik voor

het feit dat ik alles heerlijk met je heb kunnen delen! Op de één of andere manier zorg je ervoor dat alle drukte acuut van me afvalt als je in mijn buurt bent (en dat is niet omdat je

zelf zo’n rust in huis bent...!) Lievie, we gaan een spannende tijd tegemoet met lots of

changes en ik zie er ontzettend naar uit om daar samen iets moois van te maken!

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Curriculum VitaeRichard Kemper was born on January 22, 1971 in Kampen, the Netherlands. He completedhis highschool (VWO) education in 1989 at the ‘Almere college’ in Kampen. He studied

Medical Biology at the University of Groningen and graduated in 1995. In the same year he

started the Ph.D. study presented in this thesis at the department of Anaesthesiology, whichwas carried out at the department of Biological Psychiatry of the University Hospital

Groningen. Since July 1999 he is working as Clinical Research Associate at Knoll BV, BASF

Pharma, posted by Parexel Mirai BV, Amsterdam.

Full Papers• R.H.A. Kemper, M.B. Spoelstra, W.J. Meijler, G.J. Ter Horst. LPS induced hyperalgesia of intracranial

capsaicin sensitive afferents in conscious rats. Pain 78/3 (1998) 181-190.• R.H.A. Kemper, W.J. Meijler, G.J. Ter Horst. Trigeminovascular stimulation in conscious rats.

Neuroreport 8 (1997) 1123-1126.• H.J. Krugers, R.H.A. Kemper, J. Korf, G.J. Ter Horst, S. Knollema. Metyrapone reduces rat brain

damage and seizures after hypoxia-ischemia: an effect independent of modulation of plasmacorticosterone levels? Journal of Cerebral Blood Flow and Metabolism 18 (1998) 386-390.

• W.A. Kaptein, R.H.A. Kemper, K. Venema, R.G. Tiessen, J. Korf. Methodological aspects of glucosemonitoring with a slow continuous subcutaneous and intravenous ultrafiltration in rats. Biosensors &Bioelectronics 12/9-10 (1997) 967-976.

• S. Knollema, R.H.A. Kemper, J. Korf, A. Wiersma, G.J. Ter Horst, H.J. Krugers. The number ofinsults and the cerebral damage after hypoxia/ischemia are altered after acute pretreatment withcorticosterone and metyrapone. Neuroscience Research Communications 21/3 (1997) 203-211.

• W.J. Drijfhout, R.H.A. Kemper, P. Meerlo, J.M. Koolhaas, C.J. Grol, B.H.C. Westerink. A telemetrystudy on the chronic effects of microdialysis probe implantation on the activity pattern andtemperature rhythm of the rat. Journal of Neuroscience Methods 61/1-2 (1995) 191-196.

• H.J. Krugers, S. Knollema, R.H.A Kemper, G.J. Ter Horst, J. Korf. Down regulation of thehypothalamo- pituitary-adrenal axis reduces brain damage and number of seizures followinghypoxia/ischeamia in rats. Brain Research 690 (1995) 41-47.

• R.H.A. Kemper, W.J. Meijler, J. Korf, G.J. Ter Horst. Immunesystem function in migraine.(submitted)

• R.H.A. Kemper, M. Jeuring W.J. Meijler, J. Korf, G.J. Ter Horst. Intracisternally applied octreotidedoes not ameliorate orthodromic trigeminovascular nociception. (submitted)

Abstracts• R.H.A. Kemper, M.B. Spoelstra, W.J. Meijler, G.J. Ter Horst. LPS induces hyperalgesia of intracranial

capsaicin sensitive afferents in conscious rats. Soc. Neurosci. Abstr., Vol 24 Part 1, (1998) 880.• R.H.A. Kemper, M.B. Spoelstra, W.J. Meijler, G.J. Ter Horst. Central c-fos expression pattern

induced by trigeminovascular stimulation in conscious rats. Cephalalgia 17 (1997) 382.• R.H.A. Kemper, M.B. Spoelstra, L. Vosmeijer, M.G. Postma, M.J.L. De Jongste, W.J. Meijler, G.J. Ter

Horst. Dose dependent changes in behaviour and trigeminal c-fos expression after intracisternalcapsaicin infusion. Soc. Neurosci. Abstr., Vol 22 Part 2, (1996) 870.

• R.H.A. Kemper, S. Knollema, G.J. Ter Horst, J. Korf, H.J. Krugers. Glucocorticoid effects on epilepticseizures and brain damage after hypoxia/ischemia. Soc. Neurosci. Abstr., Vol 19 (1994).

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